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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 February 2012 and 10 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Range-finding test

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.4 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:

Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Experimental Preparation

An amount of test item (200 mg) was added to the surface of 2 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (1 litre) of WAF was inoculated with algal suspension (6.2 ml) to give the required test concentration of 100 mg/l loading rate WAF.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Total Organic Carbon (TOC) analysis was performed on the test preparations prepared with the omission of algal cells at 0 and 72 hours (see Appendix 4).

Observations on the test media were carried out during the mixing and testing of the WAFs.

At both the start and end of the mixing period and following a 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item floating at the media surface. Microscopic examination of the WAF showed there to be no micro-dispersions or particles of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10E+4 – 10E+5 cells/ml.


- Culturing media and conditions:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l


The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.



- Any deformed or abnormal cells observed:

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.6 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

The pH of the control and 100 mg/l loading rate WAF was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.
Dissolved oxygen:
Not applicable
Salinity:
freshwater used
Nominal and measured concentrations:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
Details on test conditions:
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 8.01 x 10E+05 cells per ml. Inoculation of 1 litre of test medium with 6.2 ml of this algal suspension gave an initial nominal cell density of 5 x 10E+03 cells per ml and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.



EFFECT PARAMETERS MEASURED :

Determination of ECx values

EL*x values were determined by inspection of the growth rate and yield data after 72 hours.

VALIDATION:

The results of the test are considered valid if the following performance criteria are met:

i) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
iii)The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: NA
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: NA
Details on results:
- Exponential growth in the control (for algal test): yes


Observations on cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatusto the test material during the range-finding test are given in Table 1.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 83 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.70 x 10E+03 cells per ml
Mean cell density of control at 72 hours : 4.71 x 10E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P>0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield

EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as for the growth rate. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P>0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.


*EL = Effective Loading Rate

Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/l, 95% confidence limits 1.2 – 1.7 mg/l
EyC50 (0 – 72 h) : 0.59 mg/l, 95% confidence limits 0.53 – 0.65 mg/l

No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.70E+03

1.11E+06

-

-

 

R2

7.00E+03

1.13E+06

 

Mean

6.35E+03

1.12E+06

10

R1

4.94E+03

1.21E+06

[6]

[8]

 

R2

4.90E+03

1.22E+06

 

Mean

4.92E+03

1.21E+06

100

R1

4.30E+03

1.21E+06

[7]

[8]

 

R2

4.88E+03

1.21E+06

 

Mean

4.59E+03

1.21E+06

 

[ ]


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.6

7.34E+03

1.98E+04

7.66E+04

3.99E+05

8.1

 

R2

5.34E+03

2.01E+04

9.03E+04

3.72E+05

 

R3

4.91E+03

2.12E+04

1.02E+05

5.46E+05

 

R4

6.34E+03

1.93E+04

8.04E+04

3.97E+05

 

R5

5.33E+03

1.96E+04

9.77E+04

4.77E+05

 

R6

4.93E+03

2.16E+04

1.11E+05

6.33E+05

 

Mean

5.70E+03

2.03E+04

9.31E+04

4.71E+05

100

R1

7.5

6.30E+03

2.03E+04

9.53E+04

4.81E+05

8.0

 

R2

4.85E+03

1.56E+04

9.52E+04

4.30E+05

 

R3

4.71E+03

2.00E+04

9.49E+04

4.33E+05

 

R4

5.55E+03

1.78E+04

9.41E+04

4.47E+05

 

R5

5.15E+03

1.81E+04

9.81E+04

4.74E+05

 

R6

5.06E+03

2.02E+04

9.66E+04

4.76E+05

 

Mean

5.27E+03

1.87E+04

9.57E+04

4.57E+05

 


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.057

0.056

0.069

 

R2

0.058

0.063

0.059

 

R3

0.060

0.066

0.070

 

R4

0.056

0.059

0.067

 

R5

0.057

0.067

0.066

 

R6

0.061

0.068

0.072

 

Mean

0.058

0.063

0.067

 


R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.061

 

3.92E+05

 

 

R2

0.060

 

3.67E+05

 

 

R3

0.065

 

5.41E+05

 

 

R4

0.061

-

3.91E+05

-

 

R5

0.063

 

4.71E+05

 

 

R6

0.067

 

6.28E+05

 

 

Mean

0.063

 

4.65E+05

 

 

SD

0.003

 

1.03E+05

 

100

R1

0.063

0

4.75E+05

 

 

R2

0.062

2

4.26E+05

 

 

R3

0.062

2

4.28E+05

 

 

R4

0.062

2

4.42E+05

 

 

R5

0.063

0

4.69E+05

 

 

R6

0.063

0

4.71E+05

 

 

Mean

0.063

1

4.52E+05

3

 

SD

0.001

 

2.25E+04

 

 


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Media Column (cm)

9.5

11.5

9.5

11.5

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present


*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effective Loading Rate
Executive summary:

Introduction.A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Methods. Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results.Exposure ofPseudokirchneriella subcapitatato the test item gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations of less than the limit of quantitation (LOQ) were obtained which was considered to be 1.0 mg C/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 11 August 2011 and 26 August 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:

- Concentrations:
100 mg/l loading rate WAF (6 replicates)

- Sampling method:
Total Organic Carbon (TOC) analysis was performed on the test preparations prepared with the omission of algal cells at 0 and 72 hours (see details on analytical methods section).

- Sample storage conditions before analysis:
Duplicate samples were taken and stored frozen (approximately -20DegC) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see Appendix 3 in any other in fromation on results including tables section).


Range-finding test
Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.4 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron(R) Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

PREPARATION AND APPLICATION OF TEST SOLUTION FOR THE DEFINITIVE TEST
- Method:
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

An amount of test item (200 mg) was weighed onto a glass slide and suspended within 2 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (4.3 ml) to give the required test concentration of 100 mg/l loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test preparations prepared with the omission of algal cells at 0 and 72 hours.

- Eluate:
Not applicable

- Controls:
A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.The positive control was conducted between 31 January 2011 and 10 February 2011.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.



- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
At both the start and end of the mixing period and following a 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:

TEST ORGANISM
- Common name:
The test was carried out using Pseudokirchneriella subcapitata
Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems.

- Strain:
strain CCAP 278/4

- Source:
Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined below:
Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1DegC.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+03 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1DegC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.


ACCLIMATION

- Acclimation period:
Not recorded.



- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:

Not applicable
Hardness:

Not recorded.
Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH of the control and 100 mg/l loading rate WAF test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.
Dissolved oxygen:

Not recorded.
Salinity:

freshwater used
Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.
Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml
- Aeration: No aeration

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.65E+05 cells per ml. Inoculation of 1 litre of test medium with 11 ml of this algal suspension gave an initial nominal cell density of 4E+03 cells per ml and had no significant dilution effect on the final test concentration.

- Control end cells density: algal cell density was approximately 7.67 E+05 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 4E+03 cells/ml.

- No. of vessels per concentration (replicates):
six replicate flasks .

- No. of vessels per control (replicates):
Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes



OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :

As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.17 E+06 cells per ml. Inoculation of 1 litre of test medium with 4.3 ml of this algal suspension gave an initial nominal cell density of 5E+03 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hour


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS).
EL*x values were determined by inspection of the growth rate and yield data after 72 hours.
*EL = Effective Loading Rate



- Chlorophyll measurement:
Not recorded


VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: none specified
Details on results:

Validation of Mixing Period
Pre-study work (see Appendix 3 in any other inforamtion on results including tables section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.
Therefore the test item was prepared as a Water Accommodate fraction using a 23-Hour stirring period followed by a 1-Hour standing period.

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 in any other infromation on results including tables section.
The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.
Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2in any other infromation on results including tables section . Daily specific growth rates for the control cultures are given in Table 3in any other infromation on results including tables section . Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4in any other infromation on results including tables section .
The mean cell densities versus time for the definitive test are attached in Figure 1.


Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 151 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 5.09+E03 cells per ml
Mean cell density of control at 72 hours : 7.67+ E05 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
of growth values from the exposure of Desmodesmus subspicatusto the test material during the range-finding test are given in Table1 in any other infomration on results including tables section.
The results showed no effect on growth at 10 mg/l loading rate WAF. However, growth was observed to be reduced at 100 mg/l loading rate WAF.
Based on this information loading rates of 10, 20, 40, 80 and 160 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.



Growth data
From the data given in Tables 2 and 4 in anyn other information on results including tables section, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where ErL*x is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P equal to or greather than 0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.
Inhibition of yield
EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where EyL*x is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Physico-chemical measurements
The pH values of the control and 100 mg/l loading rate WAF test concentration are given in Table 2 in any other information on results including tables section. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH value of the control cultures (see Table 2 in any other information on results including tables section) was observed to increase from pH 8.0 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface (see Table 5 in any other information on results including tables section).
Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the WAFs containing no algal cells was conducted at 0 and 72 hours (see Appendix 4) and showed measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l, were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.





Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/l
EyC50 (0 – 72 h) : 0.48 mg/l, 95% confidence limits 0.43 – 0.54 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.59E+03

8.51E+05

-

-

 

R2

5.90E+03

9.78E+05

 

Mean

5.75E+03

9.14E+05

10

R1

5.30E+03

7.84E+05

1

14

 

R2

5.52E+03

7.89E+05

 

Mean

5.41E+03

7.87E+05

100

R1

5.31E+03

1.14E+06

[3]

[11]

 

R2

5.77E+03

8.82E+05

 

Mean

5.54E+03

1.01E+06

 


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

8.0

5.13E+03

3.05E+04

1.71E+05

8.79E+05

8.1

 

R2

5.12E+03

2.79E+04

1.46E+05

8.89E+05

 

R3

5.06E+03

2.91E+04

1.63E+05

7.58E+05

 

R4

5.10E+03

2.84E+04

1.47E+05

6.55E+05

 

R5

5.07E+03

2.57E+04

1.57E+05

7.12E+05

 

R6

5.06E+03

2.88E+04

1.61E+05

7.05E+05

 

Mean

5.09E+03

2.84E+04

1.57E+05

7.67E+05

100

R1

7.9

5.18E+03

3.05E+04

1.78E+05

8.97E+05

8.0

 

R2

5.06E+03

2.96E+04

1.57E+05

7.78E+05

 

R3

5.06E+03

2.48E+04

1.30E+05

6.90E+05

 

R4

5.00E+03

3.20E+04

1.66E+05

8.88E+05

 

R5

5.09E+03

2.97E+04

1.54E+05

8.17E+05

 

R6

4.76E+03

2.60E+04

1.55E+05

7.87E+05

 

Mean

5.02E+03

2.88E+04

1.57E+05

8.10E+05


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.075

0.072

0.068

 

R2

0.072

0.069

0.075

 

R3

0.073

0.072

0.064

 

R4

0.072

0.069

0.062

 

R5

0.068

0.075

0.063

 

R6

0.073

0.072

0.062

 

Mean

0.072

0.072

0.066

 


R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate Yield in the Definitive Test

Nominal Loading Rate
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.072

 

8.74E+05

 

 

R2

0.072

 

8.84E+05

 

 

R3

0.070

 

7.53E+05

 

 

R4

0.068

-

6.50E+05

-

 

R5

0.069

 

7.07E+05

 

 

R6

0.069

 

7.00E+05

 

 

Mean

0.070

 

7.62E+05

 

 

SD

0.002

 

9.68E+04

 

100

R1

0.072

[3]

8.92E+05

 

 

R2

0.070

0

7.73E+05

 

 

R3

0.068

3

6.85E+05

 

 

R4

0.072

[3]

8.83E+05

 

 

R5

0.071

[1]

8.12E+05

 

 

R6

0.070

0

7.82E+05

 

 

Mean

0.071

[1]

8.04E+05

[6]

 

SD

0.002

 

7.69E+04

 


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Media Column (cm)

12

12

12

12

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

 


*= Start of mixing period

+= End of mixing period

Appendix3      Validation of Mixing Period

Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic items, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.

The results are summarised as follows:

Nominal
Loading Rate

(mg/l)

Time (Hours)

24

96

mg C/l

mg C/l Corrected for Control

mg C/l

mg C/l Corrected for Control

Control

<LOQ

-

<LOQ

-

100

4.89

4.89

4.27

4.27

It is evident from this work that increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.*


LOQ = limit of quantitation which was considered to be 1.0 mg C/l.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effect Loading Rate
Executive summary:

Introduction.A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods. Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results and Conclusion.Exposure ofPseudokirchneriella subcapitatato the test item gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l, were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2009 - 28 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and nominal 100 mg test item/L
- Sampling method: Duplicate samples from the freshly prepared test media (without algae) and the control were taken at the start of the test. Duplicate samples from the only concentration and the control were taken at the end of the test. For the control, only one of the duplicate samples was analysed from each of the sampling times.
- Sample storage conditions before analysis: Samples taken at the start of the test were stored in a freezer (< - 10 °C), protected from the light. Samples taken at the end of the test were not stored and analysed directly.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg of the test item was added directly to 2000 mL of the test media and slightly stirred for about 24 hours at room temperature in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium.
- Differential loading: None
- Controls: Untreated test water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations, clear test medium
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): “Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Gӧttingen”, 37073 Gӧttingen, Germany
- Age of inoculum (at test initiation): Cells were taken from an exponentially growing pre-culture set up 4 days prior to the test start.
- Method of cultivation: Under standardised conditions according to test guidelines

ACCLIMATION
- Acclimation period: Cells were taken from an exponentially growing pre-culture set up 4 days prior to the test start.
- Culturing media and conditions (same as test or not): Cultivated in the laboratory under standardised conditions according to the test guidelines
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
75 h
Hardness:
0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
23 °C
pH:
Test start (0 hours): 8.1
Test end (72 hours): 7.9 - 8.0
Dissolved oxygen:
Not reported; media continuously stirred
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 100 mg test item/L
Measured: < LOQ
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): covered with glass dishes
- Material, size, headspace, fill volume: 50 mL flasks containing approx. 50 mL of test medium
- Aeration: Continuously stirred
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 5000 cells per mL
- Control end cells density: mean; 41.047 x 10^4 mL; standard deviation; 8.338 x 10^4 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD medium) prepared according to guideline.
- Total organic carbon: < LOQ
- Particulate matter: Not reported
- Conductivity: < 5 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH measured in all test concentrations and the control at the start and end of the test. Temperature measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: None
- Photoperiod: Continuous
- Light intensity and quality: Mean: 6720 Lux (range: 6250 – 7100 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth rate and yield at 72 hours
- Determination of cell concentrations: Determined by spectrophotometer at 24, 48 and 72 hours
- Chlorophyll measurement: None

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None; Limit test performed
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: Range finding study conducted but the concentrations are not reported. The study states concentrations above the limit concentration (100 mg/L or limit of solubility) were not tested in accordance with guidelines.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities observed microscopically
- Unusual cell shape: None reported
- Colour differences: None
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Other: The validity criteria were met as:
Increase in cell density by at least a factor of 16 within 72 hours in the control cultures (observed; 82.1-fold increase)
The coefficient of variation of sectional (daily) growth rates in control cultures must not exceed 35 % (observed; 24 %)
The coefficient of variation of average growth rates between control replicates must not exceed 7 % (observed; 5 %)
- Any stimulation of growth found in any treatment: Stimulation observed in the 100 mg/L test concentration, the only concentration tested
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: No effects observed
Results with reference substance (positive control):
Reference substance results not reported
Reported statistics and error estimates:
The test item growth and yield values were compared to the control with a Student t-test (α = 0.05, one-sided). The NOEC and LOEC were calculated using appropriate statistical methods

Table 1: Influenece of the test item on the Growth Pseudokirchneriella subcapitata

 Parameter (0 - 72 hours)  Growth rate (mg test item/L)  Yield (mg test item/L)
 72 -hour EC50  > 100 (nominal)  > 100 (nominal)
 95 % conf. limits  n.d.  n.d.
 72 -hour EC10  > 100 (nominal)  > 100 (nominal)
 95 % conf. limits  n.d.  n.d.
 72 -hour NOEC   100 (nominal)   100 (nominal)
 72 -hour LOEC  > 100 (nominal)  > 100 (nominal)

n.d. not determinable

Table 2: Algal Cell Densities During the Test Period of 72 Hours

Nominal Concentration (mg test item/L)

 Flask No.

 Density of algal cells (10^4/mL) after

 24 hours

 48 hours

 72 hours

 Control

 1

 1.384

 10.128

 49.948

 2  1.519  7.841  48.199
 3  1.653  7.303  41.742
 4  1.519  6.362  34.478
 5  1.519  5.285  28.155
 6  1.384  7.438  43.760
 m  1.496  7.393  41.047
 s  0.101  1.625  8.338
 100  1  1.922  7.572  53.850
 2  1.788  4.344  44.836
 3  1.519  4.882  40.531
 4  1.250  9.187  52.773
 5  1.250  12.012  57.347
 6  1.519  9.187  38.783
 m  1.541  7.864  48.020
 s  0.275  2.901  7.683

m: mean value; s: standard deviation

At test start 5000 algal cells/mL were incubated

Table 3: Growth Rates µ and Percentage Inhibition of µ During the Test Period

Nominal Concentration (mg/L)  Growth rates µ (1/day) and % inhibition of µ    
 0 - 24 hours  0 - 48 hours  0 - 72 hours
 µ  %    µ  %    µ  %  
 Control

 1.094

 0.0

 

 1.337

 0.0

 

 1.463

 0.0

 

 100

 1.112

 -1.7

 -

 1.347

 -0.7

 -

 1.518

 -3.8

 -

negative values in '% inhibition' indicate an increase in growth relative to that of the control

- no significant difference compared to the control (tested with Student t-test, α = 0.05, one-sided)

Table 4: Yield, y, and Percentage Inhibition of y During the Test Period

Nominal Concentration (mg/L)

 Yield y (x*10^4 cells/mL) and % inhibition of y    
 0 - 24 hours  0 - 48 hours  0 - 72 hours
 y  %    y  %    y  %  
 Control  0.996  0.0    6.893  0.0    40.547  0.0  
 100  1.041  -4.5  -  7.364  -6.8  -  47.250  -17.2  -

negative values in '% inhibition' indicate an increase in growth relative to that of the control

- no significant difference compared to the control (tested with Student t-test,α= 0.05, one-sided)

Table 5: Appearance of the Test Item in Test medium

Nominal Concetration (mg/L)    

Appearance of the test item in the test medium

Start (0 hours)

 24 hours

 48 hours

 End (72 hours)

 100

 0

 0

 0

 0

0 - No remarkable observations, clear test medium

Table 6: pH-Values in the Test Media at the Start and the End of the Test

Nominal Concentration

 pH value

 (mg test item/L)

 Start (0 hours)

 End (72 hours)

 Control

 8.1

 7.9

 100

 8.1

 8.0

Table 7: Temperature in the Test Medium During the Test Period

    

Water Temperature (°C)

Start (0 hours)

 24 hours

 48 hours

 End (72 hours)

 Control

 23

 23

 23

 23

Table 8: Results for the Determination of the Test Item in the Test Samples

 Sample Description

 Total Carbon

 Inorganic Carbon 

 Total Organic Carbon (TOC)

 mg/L

 age (h)

 Found (mg/L)

 DF

 Calculated (mg Carbon/L)1

 Found (mg/l)

 DF

 Calculated (mg/Carbon/l)1

 (mg Carbon/L)

 Control

 0

 4.94

 1

 4.94

 5.72

 1

 5.72

 <LOQ

 Control

 0

 4.82

 1

 4.82

 6.53

 1

 6.53

  <LOQ

100

 0

 4.73

 1

 4.73

 5.50

 1

 5.50

  <LOQ

100

 0

 4.62

 1

 4.62

 5.47

 1

 5.47

  <LOQ

 100

 72

 4.61

 1

 4.61

 6.10

 1

 6.10

  <LOQ

 100

 72

 4.55

 1

 4.55

 6.21

 1

 6.21

  <LOQ

1The tabulated results represent rounded results calculated on the exact raw data

LOQ: Limit of Quantification = 3 mg Carbon/L

D.F. Dilution Factor

Validity criteria fulfilled:
yes
Remarks:
Increase in cell density observed: 82.1-fold increase. The coefficient of variation of sectional growth rates in control observed: 24 %. Coefficient of variation of average growth rates between control replicates observed: 5 %
Conclusions:
The toxicity of the test item to the green algae Pseudokirchneriella subcapitata in 72-hour limit test was investigated. Based on nominal concentrations the EC10 and EC50 for growth rate and yield were determined to be >100 mg/L. The NOEC and LOEC were determined to be ≥100 and >100 mg/L, respectively.
Executive summary:

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was investigated in 72-hour limit test was according to the OECD 201 guideline. The study was conducted as a limit test using a water accommodated fraction due to the test item being a complex UVCB. The test item was prepared as a water accommodated fraction by adding 200 mg of the test item to 2000 mL of test water and slightly stirring for 24 hours; the aqueous phase was then drawn off and used in the test. A control, containing test water only, was conducted in parallel. Six replicates of the control and test item were performed. Analysis of the test item concentrations was performed by calibrated TOC analysis at test start and test end. The cell density all solutions was determined at 24, 48 and 72 hours, and percentage inhibition of growth rate and yield was determined at 72 hours.

Based on nominal concentrations the EC10 and EC50 for growth rate and yield were determined to be >100 mg/L, the only concentration tested. The NOEC and LOEC were determined to be ≥100 and >100 mg/L, respectively.

The study is a GLP compliant guideline experimental study fully acceptable for assessment of this endpoint.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
other information
Study period:
18 August 2011 and 13 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Range-finding test

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rates to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Amounts of test item (20 and 200 mg) were separately weighed onto glass slides and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.3 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Vehicle:
no
Details on test solutions:
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.

Amounts of test item (12.5, 25, 50, 100 and 200 mg) were each separately weighed onto glass slides and suspended within 2 litres of culture medium to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (5.0 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l loading rate WAF.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Total organic carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control and each loading rate WAF at 0 and 72 hours for this analysis (see Appendix 4). Duplicate samples were taken and stored frozen (approximately -20ºC) for further analysis if necessary.

Evidence of undissolved material:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following a 1-Hour standing period all loading rate WAFs were observed to have formed clear colourless media columns with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAFs showed there to be no globules or micro-dispersions of test item present.
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 6.25, 12.5 and 25 mg/l loading rate WAF test cultures were observed to be green dispersions. The 50 mg/l loading rate WAF test cultures were observed to be very pale green dispersions whilst the 100 mg/l loading rate WAF test cultures were observed to be clear colourless solutions.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test Species
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.

Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


- Any deformed or abnormal cells observed:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/l loading rate WAF, however cell debris was observed to be present in the test cultures at 100 mg/l loading rate WAF.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH values of the control and each test preparation are given in Table 2 - see section any other inforamtion on results.

The pH value of the control cultures (see Table 2 in section any other information on results) was determined to be pH 8.1 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Dissolved oxygen:
Not applicable
Salinity:
freshwater used
Nominal and measured concentrations:
The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.
Details on test conditions:
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.96 x 10^5 cells per ml. Inoculation of 500 ml of test medium with 5.0 ml of this algal suspension gave an initial nominal cell density of 5 x 10^03 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.


- Control end cells density: algal cell density was approximately 7.05E+05 cells/ml


OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- Light intensity and quality:
warm white lighting (380 – 730 nm)


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Determination of ECx values

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). EL*x values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EL*50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
* The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

* The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

* The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits 50 - 53 mg/l
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% confidence limits 32 - 39 mg/l
Details on results:
Exponential growth in the control (for algal test): yes

Observations on cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/l loading rate WAF, however cell debris was observed to be present in the test cultures at 100 mg/l loading rate WAF.


Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata the test material during the range-finding test are given in Table1.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 122 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.77E+03 cells per ml
Mean cell density of control at 72 hours : 7.05E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 18% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErL*10 (0 - 72 h) : 46 mg/l loading rate WAF
ErL*20 (0 - 72 h) : 48 mg/l loading rate WAF
ErL*50 (0 - 72 h) : 52 mg/l loading rate WAF; 95% confidence limits 50 - 53 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/l loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 25 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 50 mg/l loading rate WAF.

Inhibition of yield

EyL*10 (0 - 72 h) : 23 mg/l loading rate WAF
EyL*20 (0 - 72 h) : 27 mg/l loading rate WAF
EyL*50 (0 - 72 h) : 36 mg/l loading rate WAF; 95% confidence limits 32 - 39 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as for the frath rate data. There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/l loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 25 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 50 mg/l loading rate WAF.

EL = Effective Loading Rate


Inhibition of yield


EyL*10(0 - 72 h)           : 14 mg/l loading rate WAF
EyL*20(0 - 72 h)           : 17 mg/l loading rate WAF
EyL*50(0 - 72 h)           : 29 mg/l loading rate WAF; 95% confidence limits 25 – 34 mg/l loading rate WAF
where EyL*xis the loading rate that reduced yield by x%.

There were no statistically significant differences between the control and 10 mg/l loading rate WAF (P³0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 10 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 20 mg/l loading rate WAF.


 Inhibition of biomass integral
EbL*10(0 - 72 h)           : 12 mg/l loading rate WAF
EbL*20(0 - 72 h)           : 16 mg/l loading rate WAF
EbL*50(0 - 72 h)           : 30 mg/l loading rate WAF; 95% confidence limits 26 – 34 mg/l loading rate WAF
where EbL*xis the loading rate that reduced biomass integral by x%.

 There were no statistically significant differences between the control and 10 mg/l loading rate WAF (P³0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on biomass integral was 10 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on biomass integral was 20 mg/l loading rate WAF.

*EL = Effective Loading Rate
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/l
EyC50 (0 – 72 h) : 0.48 mg/l, 95% confidence limits 0.43 – 0.54 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table 1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.46E+03

1.09E+06

-

-

 

R2

5.14E+03

1.07E+06

 

Mean

5.30E+03

1.08E+06

10

R1

5.09E+03

1.29E+06

[4]

[18]

 

R2

5.06E+03

1.28E+06

 

Mean

5.08E+03

1.28E+06

100

R1

5.62E+03

8.07E+03

93

100

 

R2

5.00E+03

7.66E+03

 

Mean

5.31E+03

7.86E+03

 


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

8.1

5.11E+03

1.93E+04

1.05E+05

9.43E+05

8.1

 

R2

5.03E+03

2.23E+04

1.25E+05

5.09E+05

 

R3

5.01E+03

1.94E+04

1.20E+05

5.77E+05

 

R4

9.43E+03

6.46E+04

3.06E+05

9.70E+05

 

R5

5.02E+03

2.43E+04

1.32E+05

6.46E+05

 

R6

5.03E+03

2.39E+04

1.04E+05

5.84E+05

 

Mean

5.77E+03

2.90E+04

1.49E+05

7.05E+05

6.25

R1

8.0

6.02E+03

3.54E+04

2.10E+05

8.23E+05

8.0

 

R2

5.09E+03

2.49E+04

1.27E+05

6.33E+05

 

R3

5.18E+03

2.17E+04

1.22E+05

4.40E+05

 

Mean

5.43E+03

2.73E+04

1.53E+05

6.32E+05

12.5

R1

7.8

5.11E+03

3.34E+04

1.73E+05

8.95E+05

7.9

 

R2

5.13E+03

2.44E+04

1.35E+05

5.58E+05

 

R3

5.04E+03

2.32E+04

1.24E+05

6.32E+05

 

Mean

5.09E+03

2.70E+04

1.44E+05

6.95E+05

25

R1

7.7

5.10E+03

3.00E+04

1.05E+05

6.38E+05

7.9

 

R2

5.09E+03

2.38E+04

1.39E+05

6.48E+05

 

R3

5.02E+03

2.11E+04

1.24E+05

5.05E+05

 

Mean

5.07E+03

2.50E+04

1.23E+05

5.97E+05

50

R1

7.7

5.53E+03

6.36E+03

2.74E+04

1.03E+05

7.9

 

R2

5.06E+03

5.35E+03

2.59E+04

1.29E+05

 

R3

5.20E+03

4.82E+03

2.90E+04

1.11E+05

 

Mean

5.26E+03

5.51E+03

2.74E+04

1.14E+05

100

R1

7.7

6.33E+03

1.45E+03

2.07E+03

5.58E+03

7.8

 

R2

5.05E+03

2.74E+03

1.44E+03

3.13E+03

 

R3

5.06E+03

2.05E+03

4.78E+03

2.00E+03

 

Mean

5.48E+03

2.08E+03

2.77E+03

3.57E+03


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.056

0.071

0.091

 

R2

0.062

0.072

0.059

 

R3

0.056

0.076

0.065

 

R4

0.107

0.065

0.048

 

R5

0.066

0.071

0.066

 

R6

0.065

0.061

0.072

 

Mean

0.069

0.069

0.067


R1- R6= Replicates 1 to 6

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:


Response Variable EL*50 95% Confidence Limits No Observed Effect Lowest Observed Effect
(mg/l Loading rate) (mg/l Loading Rate WAF) Loading Rate (NOEL) (mg/l) Loading Rate (LOEL) (mg/l)
Growth Rate 52 50 - 53 25 50
Yield 36 32 - 39 25 50
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable

EL*50
(mg/l Loading Rate WAF)

95% Confidence Limits (mg/l Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/l)

Lowest Observed Effect Loading Rate (LOEL) (mg/l)

Growth Rate

52

50

-

53

25

50

Yield

36

32

-

39

25

50

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given that measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l were obtained for the control and all test loading rates, it was considered that the results gave no evidence of the presence of test item in the WAF.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
18 August 2011 and 13 September 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13 of IUCLID.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Range-finding test

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rates to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Amounts of test item (20 and 200 mg) were separately weighed onto glass slides and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.3 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Vehicle:
no
Details on test solutions:
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.

Amounts of test item (12.5, 25, 50, 100 and 200 mg) were each separately weighed onto glass slides and suspended within 2 litres of culture medium to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 6.25, 12.5, 25, 50 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (5.0 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l loading rate WAF.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Total organic carbon analysis
Analysis of the WAFs was carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control and each loading rate WAF at 0 and 72 hours for this analysis (see Appendix 4). Duplicate samples were taken and stored frozen (approximately -20ºC) for further analysis if necessary.

Evidence of undissolved material:
Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period and following a 1-Hour standing period all loading rate WAFs were observed to have formed clear colourless media columns with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAFs showed there to be no globules or micro-dispersions of test item present.
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control, 6.25, 12.5 and 25 mg/l loading rate WAF test cultures were observed to be green dispersions. The 50 mg/l loading rate WAF test cultures were observed to be very pale green dispersions whilst the 100 mg/l loading rate WAF test cultures were observed to be clear colourless solutions.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test Species
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.

Culturing media and conditions:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/l of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


- Any deformed or abnormal cells observed:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/l loading rate WAF, however cell debris was observed to be present in the test cultures at 100 mg/l loading rate WAF.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH values of the control and each test preparation are given in Table 2 - see section any other inforamtion on results.

The pH value of the control cultures (see Table 2 in section any other information on results) was determined to be pH 8.1 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Dissolved oxygen:
Not applicable
Salinity:
freshwater used
Nominal and measured concentrations:
The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.
Details on test conditions:
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.96 x 10^5 cells per ml. Inoculation of 500 ml of test medium with 5.0 ml of this algal suspension gave an initial nominal cell density of 5 x 10^03 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.


- Control end cells density: algal cell density was approximately 7.05E+05 cells/ml


OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- Light intensity and quality:
warm white lighting (380 – 730 nm)


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Determination of ECx values

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). EL*x values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EL*50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
* The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

* The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

* The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
52 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits 50 - 53 mg/l
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% confidence limits 32 - 39 mg/l
Details on results:
Exponential growth in the control (for algal test): yes

Observations on cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/l loading rate WAF, however cell debris was observed to be present in the test cultures at 100 mg/l loading rate WAF.


Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata the test material during the range-finding test are given in Table1.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 122 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.77E+03 cells per ml
Mean cell density of control at 72 hours : 7.05E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 18% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErL*10 (0 - 72 h) : 46 mg/l loading rate WAF
ErL*20 (0 - 72 h) : 48 mg/l loading rate WAF
ErL*50 (0 - 72 h) : 52 mg/l loading rate WAF; 95% confidence limits 50 - 53 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/l loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 25 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 50 mg/l loading rate WAF.

Inhibition of yield

EyL*10 (0 - 72 h) : 23 mg/l loading rate WAF
EyL*20 (0 - 72 h) : 27 mg/l loading rate WAF
EyL*50 (0 - 72 h) : 36 mg/l loading rate WAF; 95% confidence limits 32 - 39 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as for the frath rate data. There were no statistically significant differences between the control, 6.25, 12.5 and 25 mg/l loading rate WAFs (P0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 25 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 50 mg/l loading rate WAF.

EL = Effective Loading Rate


Inhibition of yield


EyL*10(0 - 72 h)           : 14 mg/l loading rate WAF
EyL*20(0 - 72 h)           : 17 mg/l loading rate WAF
EyL*50(0 - 72 h)           : 29 mg/l loading rate WAF; 95% confidence limits 25 – 34 mg/l loading rate WAF
where EyL*xis the loading rate that reduced yield by x%.

There were no statistically significant differences between the control and 10 mg/l loading rate WAF (P³0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 10 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on yield was 20 mg/l loading rate WAF.


 Inhibition of biomass integral
EbL*10(0 - 72 h)           : 12 mg/l loading rate WAF
EbL*20(0 - 72 h)           : 16 mg/l loading rate WAF
EbL*50(0 - 72 h)           : 30 mg/l loading rate WAF; 95% confidence limits 26 – 34 mg/l loading rate WAF
where EbL*xis the loading rate that reduced biomass integral by x%.

 There were no statistically significant differences between the control and 10 mg/l loading rate WAF (P³0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on biomass integral was 10 mg/l loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on biomass integral was 20 mg/l loading rate WAF.

*EL = Effective Loading Rate
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/l
EyC50 (0 – 72 h) : 0.48 mg/l, 95% confidence limits 0.43 – 0.54 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table 1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.46E+03

1.09E+06

-

-

 

R2

5.14E+03

1.07E+06

 

Mean

5.30E+03

1.08E+06

10

R1

5.09E+03

1.29E+06

[4]

[18]

 

R2

5.06E+03

1.28E+06

 

Mean

5.08E+03

1.28E+06

100

R1

5.62E+03

8.07E+03

93

100

 

R2

5.00E+03

7.66E+03

 

Mean

5.31E+03

7.86E+03

 


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

8.1

5.11E+03

1.93E+04

1.05E+05

9.43E+05

8.1

 

R2

5.03E+03

2.23E+04

1.25E+05

5.09E+05

 

R3

5.01E+03

1.94E+04

1.20E+05

5.77E+05

 

R4

9.43E+03

6.46E+04

3.06E+05

9.70E+05

 

R5

5.02E+03

2.43E+04

1.32E+05

6.46E+05

 

R6

5.03E+03

2.39E+04

1.04E+05

5.84E+05

 

Mean

5.77E+03

2.90E+04

1.49E+05

7.05E+05

6.25

R1

8.0

6.02E+03

3.54E+04

2.10E+05

8.23E+05

8.0

 

R2

5.09E+03

2.49E+04

1.27E+05

6.33E+05

 

R3

5.18E+03

2.17E+04

1.22E+05

4.40E+05

 

Mean

5.43E+03

2.73E+04

1.53E+05

6.32E+05

12.5

R1

7.8

5.11E+03

3.34E+04

1.73E+05

8.95E+05

7.9

 

R2

5.13E+03

2.44E+04

1.35E+05

5.58E+05

 

R3

5.04E+03

2.32E+04

1.24E+05

6.32E+05

 

Mean

5.09E+03

2.70E+04

1.44E+05

6.95E+05

25

R1

7.7

5.10E+03

3.00E+04

1.05E+05

6.38E+05

7.9

 

R2

5.09E+03

2.38E+04

1.39E+05

6.48E+05

 

R3

5.02E+03

2.11E+04

1.24E+05

5.05E+05

 

Mean

5.07E+03

2.50E+04

1.23E+05

5.97E+05

50

R1

7.7

5.53E+03

6.36E+03

2.74E+04

1.03E+05

7.9

 

R2

5.06E+03

5.35E+03

2.59E+04

1.29E+05

 

R3

5.20E+03

4.82E+03

2.90E+04

1.11E+05

 

Mean

5.26E+03

5.51E+03

2.74E+04

1.14E+05

100

R1

7.7

6.33E+03

1.45E+03

2.07E+03

5.58E+03

7.8

 

R2

5.05E+03

2.74E+03

1.44E+03

3.13E+03

 

R3

5.06E+03

2.05E+03

4.78E+03

2.00E+03

 

Mean

5.48E+03

2.08E+03

2.77E+03

3.57E+03


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.056

0.071

0.091

 

R2

0.062

0.072

0.059

 

R3

0.056

0.076

0.065

 

R4

0.107

0.065

0.048

 

R5

0.066

0.071

0.066

 

R6

0.065

0.061

0.072

 

Mean

0.069

0.069

0.067


R1- R6= Replicates 1 to 6

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the following results:


Response Variable EL*50 95% Confidence Limits No Observed Effect Lowest Observed Effect
(mg/l Loading rate) (mg/l Loading Rate WAF) Loading Rate (NOEL) (mg/l) Loading Rate (LOEL) (mg/l)
Growth Rate 52 50 - 53 25 50
Yield 36 32 - 39 25 50
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable

EL*50
(mg/l Loading Rate WAF)

95% Confidence Limits (mg/l Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/l)

Lowest Observed Effect Loading Rate (LOEL) (mg/l)

Growth Rate

52

50

-

53

25

50

Yield

36

32

-

39

25

50

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given that measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l were obtained for the control and all test loading rates, it was considered that the results gave no evidence of the presence of test item in the WAF.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2 February 2012 and 10 February 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Range-finding test

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (4.4 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:

Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

Experimental Preparation

An amount of test item (200 mg) was added to the surface of 2 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (1 litre) of WAF was inoculated with algal suspension (6.2 ml) to give the required test concentration of 100 mg/l loading rate WAF.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Total Organic Carbon (TOC) analysis was performed on the test preparations prepared with the omission of algal cells at 0 and 72 hours (see Appendix 4).

Observations on the test media were carried out during the mixing and testing of the WAFs.

At both the start and end of the mixing period and following a 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item floating at the media surface. Microscopic examination of the WAF showed there to be no micro-dispersions or particles of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10E+4 – 10E+5 cells/ml.


- Culturing media and conditions:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l


The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.



- Any deformed or abnormal cells observed:

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.6 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

The pH of the control and 100 mg/l loading rate WAF was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter.
Dissolved oxygen:
Not applicable
Salinity:
freshwater used
Nominal and measured concentrations:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
Details on test conditions:
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 8.01 x 10E+05 cells per ml. Inoculation of 1 litre of test medium with 6.2 ml of this algal suspension gave an initial nominal cell density of 5 x 10E+03 cells per ml and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.



EFFECT PARAMETERS MEASURED :

Determination of ECx values

EL*x values were determined by inspection of the growth rate and yield data after 72 hours.

VALIDATION:

The results of the test are considered valid if the following performance criteria are met:

i) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
iii)The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.


Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: NA
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: NA
Details on results:
- Exponential growth in the control (for algal test): yes


Observations on cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatusto the test material during the range-finding test are given in Table 1.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 83 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.70 x 10E+03 cells per ml
Mean cell density of control at 72 hours : 4.71 x 10E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 9% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.


Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P>0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield

EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as for the growth rate. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P>0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.


*EL = Effective Loading Rate

Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/l, 95% confidence limits 1.2 – 1.7 mg/l
EyC50 (0 – 72 h) : 0.59 mg/l, 95% confidence limits 0.53 – 0.65 mg/l

No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.70E+03

1.11E+06

-

-

 

R2

7.00E+03

1.13E+06

 

Mean

6.35E+03

1.12E+06

10

R1

4.94E+03

1.21E+06

[6]

[8]

 

R2

4.90E+03

1.22E+06

 

Mean

4.92E+03

1.21E+06

100

R1

4.30E+03

1.21E+06

[7]

[8]

 

R2

4.88E+03

1.21E+06

 

Mean

4.59E+03

1.21E+06

 

[ ]


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.6

7.34E+03

1.98E+04

7.66E+04

3.99E+05

8.1

 

R2

5.34E+03

2.01E+04

9.03E+04

3.72E+05

 

R3

4.91E+03

2.12E+04

1.02E+05

5.46E+05

 

R4

6.34E+03

1.93E+04

8.04E+04

3.97E+05

 

R5

5.33E+03

1.96E+04

9.77E+04

4.77E+05

 

R6

4.93E+03

2.16E+04

1.11E+05

6.33E+05

 

Mean

5.70E+03

2.03E+04

9.31E+04

4.71E+05

100

R1

7.5

6.30E+03

2.03E+04

9.53E+04

4.81E+05

8.0

 

R2

4.85E+03

1.56E+04

9.52E+04

4.30E+05

 

R3

4.71E+03

2.00E+04

9.49E+04

4.33E+05

 

R4

5.55E+03

1.78E+04

9.41E+04

4.47E+05

 

R5

5.15E+03

1.81E+04

9.81E+04

4.74E+05

 

R6

5.06E+03

2.02E+04

9.66E+04

4.76E+05

 

Mean

5.27E+03

1.87E+04

9.57E+04

4.57E+05

 


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.057

0.056

0.069

 

R2

0.058

0.063

0.059

 

R3

0.060

0.066

0.070

 

R4

0.056

0.059

0.067

 

R5

0.057

0.067

0.066

 

R6

0.061

0.068

0.072

 

Mean

0.058

0.063

0.067

 


R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.061

 

3.92E+05

 

 

R2

0.060

 

3.67E+05

 

 

R3

0.065

 

5.41E+05

 

 

R4

0.061

-

3.91E+05

-

 

R5

0.063

 

4.71E+05

 

 

R6

0.067

 

6.28E+05

 

 

Mean

0.063

 

4.65E+05

 

 

SD

0.003

 

1.03E+05

 

100

R1

0.063

0

4.75E+05

 

 

R2

0.062

2

4.26E+05

 

 

R3

0.062

2

4.28E+05

 

 

R4

0.062

2

4.42E+05

 

 

R5

0.063

0

4.69E+05

 

 

R6

0.063

0

4.71E+05

 

 

Mean

0.063

1

4.52E+05

3

 

SD

0.001

 

2.25E+04

 

 


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Media Column (cm)

9.5

11.5

9.5

11.5

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present


*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effective Loading Rate
Executive summary:

Introduction.A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Methods. Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results.Exposure ofPseudokirchneriella subcapitatato the test item gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations of less than the limit of quantitation (LOQ) were obtained which was considered to be 1.0 mg C/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 january 2012 and 3 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study reprot was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

Amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.8 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Vehicle:
no
Details on test solutions:
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

An amount of test item (200 mg) was weighed onto a glass slide and suspended within 2 litres of culture medium to give the 100 mg/l loading rate WAF. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (1 litre) of the WAF was separately inoculated with algal suspension (20.2 ml) to give the required test concentration of 100 mg/l loading rate WAF.

Samples were taken at 0, 25, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Observations on the test media were carried out during the mixing and testing of the WAFs.

At both the start and end of the mixing period and following a 1-Hour settlement period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control and 100 mg/l loading rate WAF test group at 0 and 72 hours for this analysis (see Appendix 4). Duplicate samples were taken and stored frozen (approximately -20ºC) for further analysis if necessary.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+03 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.

- Culturing media and conditions:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l


The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:

The pH values of the control and 100 mg/l loading rate WAF are given in Table 2 - see section any other information in resutls). Temperature was maintained at 24 ± 1ºC throughout the test.


The pH value of the control cultures (see Table 2) was observed to be pH 8.0 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:
Not applicable
Salinity:
Freshwater used
Nominal and measured concentrations:
The results of the range-finding test showed no effect on growth at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
Details on test conditions:
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.48 x 10E+05 cells per ml. Inoculation of 1 litre of test medium with 20.2 ml of this algal suspension gave an initial nominal cell density of 5 x 10E+03 cells per ml and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 25, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

EL*x values were determined by inspection of the growth rate and yield data after 72 hours.

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).


Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
i) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
iii) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.



Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Not applicable
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Not applicable
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: Not applicable
Details on results:
EXAMPLE
- Exponential growth in the control (for algal test): yes


Observations on cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.


Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Psudokirchneriella subcapitata to the test item during the range-finding test are given in Table1.

The results showed no effect on growth at 10 and 100 mg/l loading rate WAF. Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 39 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.17 x 10E+03 cells per ml
Mean cell density of control at 72 hours : 2.02 x 10E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant decreases in growth rate (P>0.05) between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield

EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 5.3.2.1. There were no statistically significant decreases in yield between the control and 100 mg/l loading rate WAF (P>0.05) and therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.


*EL = Effective Loading Rate
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 41104038) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/l, 95% confidence limits 1.2 – 1.7 mg/l
EyC50 (0 – 72 h) : 0.59 mg/l, 95% confidence limits 0.53 – 0.65 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

7.08E+03

8.49E+05

-

-

 

R2

6.34E+03

6.21E+05

 

Mean

6.71E+03

7.35E+05

10

R1

6.92E+03

1.11E+06

[8]

[38]

 

R2

5.71E+03

9.14E+05

 

Mean

6.32E+03

1.01E+06

100

R1

5.65E+03

1.14E+06

[11]

[45]

 

R2

5.91E+03

9.76E+05

 

Mean

5.78E+03

1.06E+06

 

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

25 h

50 h

72 h

72 h

Control

R1

8.0

5.02E+03

1.97E+04

7.45E+04

2.26E+05

8.0

 

R2

5.15E+03

1.61E+04

6.65E+04

2.02E+05

 

R3

5.00E+03

1.55E+04

5.21E+04

1.71E+05

 

R4

5.30E+03

1.53E+04

5.59E+04

1.69E+05

 

R5

5.41E+03

1.81E+04

5.99E+04

1.93E+05

 

R6

5.12E+03

1.92E+04

7.81E+04

2.52E+05

 

Mean

5.17E+03

1.73E+04

6.45E+04

2.02E+05

100

R1

7.9

5.10E+03

2.46E+04

1.09E+05

3.50E+05

7.8

 

R2

5.04E+03

1.96E+04

8.71E+04

2.42E+05

 

R3

5.03E+03

1.86E+04

8.12E+04

2.45E+05

 

R4

5.08E+03

2.11E+04

8.70E+04

2.93E+05

 

R5

5.31E+03

1.98E+04

8.29E+04

2.47E+05

 

R6

5.01E+03

1.83E+04

7.16E+04

2.17E+05

 

Mean

5.09E+03

2.03E+04

8.65E+04

2.65E+05

 

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.055

0.053

0.051

 

R2

0.047

0.057

0.050

 

R3

0.045

0.049

0.054

 

R4

0.045

0.052

0.050

 

R5

0.052

0.048

0.053

 

R6

0.054

0.056

0.053

 

Mean

0.050

0.053

0.052

 

R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.053

 

2.21E+05

 

 

R2

0.051

 

1.96E+05

 

 

R3

0.049

 

1.66E+05

 

 

R4

0.049

-

1.64E+05

-

 

R5

0.051

 

1.87E+05

 

 

R6

0.054

 

2.47E+05

 

 

Mean

0.051

 

1.97E+05

 

 

SD

0.002

 

3.23E+04

 

100

R1

0.059

[16]

3.45E+05

 

 

R2

0.054

[6]

2.37E+05

 

 

R3

0.054

[6]

2.40E+05

 

 

R4

0.057

[12]

2.88E+05

 

 

R5

0.054

[6]

2.41E+05

 

 

R6

0.052

[2]

2.12E+05

 

 

Mean

0.055

[8]

2.60E+05

[32]

 

SD

0.003

 

4.81E+04

 

[ ]


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Media Column (cm)

11.5

12

11.5

12

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

 


*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:
yes
Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effective Loading Rate
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Exposure of Pseudokirchneriella subcapitatato the test item gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured test concentrations to be below or around the limit of quantitation (LOQ) which was considered to be 1.0 mg C/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was at or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
26 January 2012 and 3 February 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study reprot was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13 of IUCLID.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.

The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.

Amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.8 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Vehicle:
no
Details on test solutions:
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

An amount of test item (200 mg) was weighed onto a glass slide and suspended within 2 litres of culture medium to give the 100 mg/l loading rate WAF. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

An aliquot (1 litre) of the WAF was separately inoculated with algal suspension (20.2 ml) to give the required test concentration of 100 mg/l loading rate WAF.

Samples were taken at 0, 25, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Observations on the test media were carried out during the mixing and testing of the WAFs.

At both the start and end of the mixing period and following a 1-Hour settlement period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be pale green dispersions.

Analysis of the WAF was carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control and 100 mg/l loading rate WAF test group at 0 and 72 hours for this analysis (see Appendix 4). Duplicate samples were taken and stored frozen (approximately -20ºC) for further analysis if necessary.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1ºC.

Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+03 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1ºC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.

- Culturing media and conditions:

The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l


The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:

The pH values of the control and 100 mg/l loading rate WAF are given in Table 2 - see section any other information in resutls). Temperature was maintained at 24 ± 1ºC throughout the test.


The pH value of the control cultures (see Table 2) was observed to be pH 8.0 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Dissolved oxygen:
Not applicable
Salinity:
Freshwater used
Nominal and measured concentrations:
The results of the range-finding test showed no effect on growth at 10 and 100 mg/l loading rate WAF.

Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
Details on test conditions:
As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.48 x 10E+05 cells per ml. Inoculation of 1 litre of test medium with 20.2 ml of this algal suspension gave an initial nominal cell density of 5 x 10E+03 cells per ml and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 25, 50 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

EL*x values were determined by inspection of the growth rate and yield data after 72 hours.

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).


Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
i) The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
iii) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.



Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Not applicable
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Not applicable
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: Not applicable
Details on results:
EXAMPLE
- Exponential growth in the control (for algal test): yes


Observations on cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.


Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Psudokirchneriella subcapitata to the test item during the range-finding test are given in Table1.

The results showed no effect on growth at 10 and 100 mg/l loading rate WAF. Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 39 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.17 x 10E+03 cells per ml
Mean cell density of control at 72 hours : 2.02 x 10E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

From the data given in Tables 2 and 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where ErL*x is the loading rate that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant decreases in growth rate (P>0.05) between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.

Inhibition of yield

EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF

where EyL*x is the loading rate that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 5.3.2.1. There were no statistically significant decreases in yield between the control and 100 mg/l loading rate WAF (P>0.05) and therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.


*EL = Effective Loading Rate
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 41104038) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/l, 95% confidence limits 1.2 – 1.7 mg/l
EyC50 (0 – 72 h) : 0.59 mg/l, 95% confidence limits 0.53 – 0.65 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.50 mg/l

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

7.08E+03

8.49E+05

-

-

 

R2

6.34E+03

6.21E+05

 

Mean

6.71E+03

7.35E+05

10

R1

6.92E+03

1.11E+06

[8]

[38]

 

R2

5.71E+03

9.14E+05

 

Mean

6.32E+03

1.01E+06

100

R1

5.65E+03

1.14E+06

[11]

[45]

 

R2

5.91E+03

9.76E+05

 

Mean

5.78E+03

1.06E+06

 

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

25 h

50 h

72 h

72 h

Control

R1

8.0

5.02E+03

1.97E+04

7.45E+04

2.26E+05

8.0

 

R2

5.15E+03

1.61E+04

6.65E+04

2.02E+05

 

R3

5.00E+03

1.55E+04

5.21E+04

1.71E+05

 

R4

5.30E+03

1.53E+04

5.59E+04

1.69E+05

 

R5

5.41E+03

1.81E+04

5.99E+04

1.93E+05

 

R6

5.12E+03

1.92E+04

7.81E+04

2.52E+05

 

Mean

5.17E+03

1.73E+04

6.45E+04

2.02E+05

100

R1

7.9

5.10E+03

2.46E+04

1.09E+05

3.50E+05

7.8

 

R2

5.04E+03

1.96E+04

8.71E+04

2.42E+05

 

R3

5.03E+03

1.86E+04

8.12E+04

2.45E+05

 

R4

5.08E+03

2.11E+04

8.70E+04

2.93E+05

 

R5

5.31E+03

1.98E+04

8.29E+04

2.47E+05

 

R6

5.01E+03

1.83E+04

7.16E+04

2.17E+05

 

Mean

5.09E+03

2.03E+04

8.65E+04

2.65E+05

 

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.055

0.053

0.051

 

R2

0.047

0.057

0.050

 

R3

0.045

0.049

0.054

 

R4

0.045

0.052

0.050

 

R5

0.052

0.048

0.053

 

R6

0.054

0.056

0.053

 

Mean

0.050

0.053

0.052

 

R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.053

 

2.21E+05

 

 

R2

0.051

 

1.96E+05

 

 

R3

0.049

 

1.66E+05

 

 

R4

0.049

-

1.64E+05

-

 

R5

0.051

 

1.87E+05

 

 

R6

0.054

 

2.47E+05

 

 

Mean

0.051

 

1.97E+05

 

 

SD

0.002

 

3.23E+04

 

100

R1

0.059

[16]

3.45E+05

 

 

R2

0.054

[6]

2.37E+05

 

 

R3

0.054

[6]

2.40E+05

 

 

R4

0.057

[12]

2.88E+05

 

 

R5

0.054

[6]

2.41E+05

 

 

R6

0.052

[2]

2.12E+05

 

 

Mean

0.055

[8]

2.60E+05

[32]

 

SD

0.003

 

4.81E+04

 

[ ]


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Media Column (cm)

11.5

12

11.5

12

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

 


*= Start of mixing period

+= End of mixing period

Validity criteria fulfilled:
yes
Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effective Loading Rate
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Exposure of Pseudokirchneriella subcapitatato the test item gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured test concentrations to be below or around the limit of quantitation (LOQ) which was considered to be 1.0 mg C/l.

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was at or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
The study was conducted between 11 August 2011 and 26 August 2011.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:

- Concentrations:
100 mg/l loading rate WAF (6 replicates)

- Sampling method:
Total Organic Carbon (TOC) analysis was performed on the test preparations prepared with the omission of algal cells at 0 and 72 hours (see details on analytical methods section).

- Sample storage conditions before analysis:
Duplicate samples were taken and stored frozen (approximately -20DegC) for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Validation of mixing period
Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis water. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis (see Appendix 3 in any other in fromation on results including tables section).


Range-finding test
Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
The loading rate to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal loading rates of 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
Amounts of test item (20 and 200 mg) were each separately weighed onto a glass slide and suspended within 2 litres of culture medium to give the 10 and 100 mg/l loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 10 and 100 mg/l loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (200 ml) of each of the loading rate WAFs was separately inoculated with algal suspension (2.4 ml) to give the required test concentrations of 10 and 100 mg/l loading rate WAF.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron(R) Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

PREPARATION AND APPLICATION OF TEST SOLUTION FOR THE DEFINITIVE TEST
- Method:
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.

An amount of test item (200 mg) was weighed onto a glass slide and suspended within 2 litres of culture medium to give the 100 mg/l loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
An aliquot (1 litre) of the WAF was inoculated with algal suspension (4.3 ml) to give the required test concentration of 100 mg/l loading rate WAF.
Total Organic Carbon (TOC) analysis was performed on the test preparations prepared with the omission of algal cells at 0 and 72 hours.

- Eluate:
Not applicable

- Controls:
A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.The positive control was conducted between 31 January 2011 and 10 February 2011.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.



- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
At both the start and end of the mixing period and following a 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless media column with test item adhered to the glass slide suspended within the media column. Microscopic examination of the WAF showed there to be no globules or micro-dispersions of test item present.

At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:

TEST ORGANISM
- Common name:
The test was carried out using Pseudokirchneriella subcapitata
Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism in freshwater ecosystems.

- Strain:
strain CCAP 278/4

- Source:
Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium is defined below:
Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1DegC.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E+03 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1DegC until the algal cell density was approximately 10E+04 – 10E+05 cells/ml.


ACCLIMATION

- Acclimation period:
Not recorded.



- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:

Not applicable
Hardness:

Not recorded.
Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH of the control and 100 mg/l loading rate WAF test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.
Dissolved oxygen:

Not recorded.
Salinity:

freshwater used
Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal loading rates of 10 and 100 mg/l
Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/l to confirm that no effect on algal growth was observed.
Details on test conditions:

TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml
- Aeration: No aeration

- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable

- Renewal rate of test solution (frequency/flow rate): Not applicable

- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 3.65E+05 cells per ml. Inoculation of 1 litre of test medium with 11 ml of this algal suspension gave an initial nominal cell density of 4E+03 cells per ml and had no significant dilution effect on the final test concentration.

- Control end cells density: algal cell density was approximately 7.67 E+05 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 4E+03 cells/ml.

- No. of vessels per concentration (replicates):
six replicate flasks .

- No. of vessels per control (replicates):
Six replicate flasks.


GROWTH MEDIUM
- Standard medium used: yes



OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :

As in the range-finding test 250 ml glass conical flasks were used. Six flasks each containing 100 ml of test preparation were used for the control and 100 mg/l loading rate WAF treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.17 E+06 cells per ml. Inoculation of 1 litre of test medium with 4.3 ml of this algal suspension gave an initial nominal cell density of 5E+03 cells per ml and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hour


EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS).
EL*x values were determined by inspection of the growth rate and yield data after 72 hours.
*EL = Effective Loading Rate



- Chlorophyll measurement:
Not recorded


VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: 95% CL not stated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks on result:
other: none specified
Details on results:

Validation of Mixing Period
Pre-study work (see Appendix 3 in any other inforamtion on results including tables section) indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours.
Therefore the test item was prepared as a Water Accommodate fraction using a 23-Hour stirring period followed by a 1-Hour standing period.

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 in any other infromation on results including tables section.
The results showed no effect on growth at 10 and 100 mg/l loading rate WAF.
Based on this information a single loading rate of six replicates of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2in any other infromation on results including tables section . Daily specific growth rates for the control cultures are given in Table 3in any other infromation on results including tables section . Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4in any other infromation on results including tables section .
The mean cell densities versus time for the definitive test are attached in Figure 1.


Validation criteria
The following data show that the cell concentration of the control cultures increased by a factor of 151 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Mean cell density of control at 0 hours : 5.09+E03 cells per ml
Mean cell density of control at 72 hours : 7.67+ E05 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.
of growth values from the exposure of Desmodesmus subspicatusto the test material during the range-finding test are given in Table1 in any other infomration on results including tables section.
The results showed no effect on growth at 10 mg/l loading rate WAF. However, growth was observed to be reduced at 100 mg/l loading rate WAF.
Based on this information loading rates of 10, 20, 40, 80 and 160 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, were selected for the definitive test.



Growth data
From the data given in Tables 2 and 4 in anyn other information on results including tables section, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErL*10 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*20 (0 - 72 h) : >100 mg/l loading rate WAF
ErL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where ErL*x is the loading rate that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 100 mg/l loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P equal to or greather than 0.05), between the control and 100 mg/l loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/l loading rate WAF.
Inhibition of yield
EyL*10 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*20 (0 - 72 h) : >100 mg/l loading rate WAF
EyL*50 (0 - 72 h) : >100 mg/l loading rate WAF
where EyL*x is the loading rate that reduced yield by x%.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences between the control and 100 mg/l loading rate WAF (P0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/l loading rate WAF.

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Physico-chemical measurements
The pH values of the control and 100 mg/l loading rate WAF test concentration are given in Table 2 in any other information on results including tables section. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH value of the control cultures (see Table 2 in any other information on results including tables section) was observed to increase from pH 8.0 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Vortex depth measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface (see Table 5 in any other information on results including tables section).
Total organic carbon analysis
Total Organic Carbon (TOC) analysis of the WAFs containing no algal cells was conducted at 0 and 72 hours (see Appendix 4) and showed measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l, were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.





Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h) : 1.2 mg/l, 95% confidence limits 0.98 – 1.4 mg/l
EyC50 (0 – 72 h) : 0.48 mg/l, 95% confidence limits 0.43 – 0.54 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/l loading rate to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.59E+03

8.51E+05

-

-

 

R2

5.90E+03

9.78E+05

 

Mean

5.75E+03

9.14E+05

10

R1

5.30E+03

7.84E+05

1

14

 

R2

5.52E+03

7.89E+05

 

Mean

5.41E+03

7.87E+05

100

R1

5.31E+03

1.14E+06

[3]

[11]

 

R2

5.77E+03

8.82E+05

 

Mean

5.54E+03

1.01E+06

 


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1and R2= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

Nominal Loading Rate

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

8.0

5.13E+03

3.05E+04

1.71E+05

8.79E+05

8.1

 

R2

5.12E+03

2.79E+04

1.46E+05

8.89E+05

 

R3

5.06E+03

2.91E+04

1.63E+05

7.58E+05

 

R4

5.10E+03

2.84E+04

1.47E+05

6.55E+05

 

R5

5.07E+03

2.57E+04

1.57E+05

7.12E+05

 

R6

5.06E+03

2.88E+04

1.61E+05

7.05E+05

 

Mean

5.09E+03

2.84E+04

1.57E+05

7.67E+05

100

R1

7.9

5.18E+03

3.05E+04

1.78E+05

8.97E+05

8.0

 

R2

5.06E+03

2.96E+04

1.57E+05

7.78E+05

 

R3

5.06E+03

2.48E+04

1.30E+05

6.90E+05

 

R4

5.00E+03

3.20E+04

1.66E+05

8.88E+05

 

R5

5.09E+03

2.97E+04

1.54E+05

8.17E+05

 

R6

4.76E+03

2.60E+04

1.55E+05

7.87E+05

 

Mean

5.02E+03

2.88E+04

1.57E+05

8.10E+05


*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/ml/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Control

R1

0.075

0.072

0.068

 

R2

0.072

0.069

0.075

 

R3

0.073

0.072

0.064

 

R4

0.072

0.069

0.062

 

R5

0.068

0.075

0.063

 

R6

0.073

0.072

0.062

 

Mean

0.072

0.072

0.066

 


R1- R6= Replicates 1 to 6

Table 4              Inhibition of Growth Rate Yield in the Definitive Test

Nominal Loading Rate
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.072

 

8.74E+05

 

 

R2

0.072

 

8.84E+05

 

 

R3

0.070

 

7.53E+05

 

 

R4

0.068

-

6.50E+05

-

 

R5

0.069

 

7.07E+05

 

 

R6

0.069

 

7.00E+05

 

 

Mean

0.070

 

7.62E+05

 

 

SD

0.002

 

9.68E+04

 

100

R1

0.072

[3]

8.92E+05

 

 

R2

0.070

0

7.73E+05

 

 

R3

0.068

3

6.85E+05

 

 

R4

0.072

[3]

8.83E+05

 

 

R5

0.071

[1]

8.12E+05

 

 

R6

0.070

0

7.82E+05

 

 

Mean

0.071

[1]

8.04E+05

[6]

 

SD

0.002

 

7.69E+04

 


*In accordance with the OECD test guideline only the mean value for yield is calculated

R1– R6= Replicates 1 to 6

SD= Standard Deviation

Table 5              Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Media Column (cm)

12

12

12

12

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

 


*= Start of mixing period

+= End of mixing period

Appendix3      Validation of Mixing Period

Pre-study work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic items, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.

The results are summarised as follows:

Nominal
Loading Rate

(mg/l)

Time (Hours)

24

96

mg C/l

mg C/l Corrected for Control

mg C/l

mg C/l Corrected for Control

Control

<LOQ

-

<LOQ

-

100

4.89

4.89

4.27

4.27

It is evident from this work that increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.*


LOQ = limit of quantitation which was considered to be 1.0 mg C/l.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL*50 values of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

*EL = Effect Loading Rate
Executive summary:

Introduction.A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 440/2008.

Methods. Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

Results and Conclusion.Exposure ofPseudokirchneriella subcapitatato the test item gave EL*50values of greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading Rate was 100 mg/l loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations of less than the limit of quantitation (LOQ), which was considered to be 1.0 mg C/l, were obtained. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.


*EL = Effective Loading Rate

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
25 August 2009 - 28 August 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
PU TDI-1 substances all contain the same core structure, with predominantly linear alkyl chains (C8 – C18) attached. The same substance can contain structures with different alkyl chain lengths, and some substances may contain small amounts of structures with cyclic groups. PU TDI-1 structures are therefore similar between all category members, and organisms will be exposed to very similar compounds. Organisms would be exposed to common structures, only differing by the length of the alkyl chain or whether cyclic groups are present. In the body, there may be metabolism of the PU TDI-1 structures, however due to the structural similarity of the parent compounds any metabolites are also likely to be similar.

Further information is available in the read-across justification document in Section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and nominal 100 mg test item/L
- Sampling method: Duplicate samples from the freshly prepared test media (without algae) and the control were taken at the start of the test. Duplicate samples from the only concentration and the control were taken at the end of the test. For the control, only one of the duplicate samples was analysed from each of the sampling times.
- Sample storage conditions before analysis: Samples taken at the start of the test were stored in a freezer (< - 10 °C), protected from the light. Samples taken at the end of the test were not stored and analysed directly.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 200 mg of the test item was added directly to 2000 mL of the test media and slightly stirred for about 24 hours at room temperature in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium.
- Differential loading: None
- Controls: Untreated test water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations, clear test medium
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): “Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Gӧttingen”, 37073 Gӧttingen, Germany
- Age of inoculum (at test initiation): Cells were taken from an exponentially growing pre-culture set up 4 days prior to the test start.
- Method of cultivation: Under standardised conditions according to test guidelines

ACCLIMATION
- Acclimation period: Cells were taken from an exponentially growing pre-culture set up 4 days prior to the test start.
- Culturing media and conditions (same as test or not): Cultivated in the laboratory under standardised conditions according to the test guidelines
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
75 h
Hardness:
0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
23 °C
pH:
Test start (0 hours): 8.1
Test end (72 hours): 7.9 - 8.0
Dissolved oxygen:
Not reported; media continuously stirred
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 100 mg test item/L
Measured: < LOQ
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): covered with glass dishes
- Material, size, headspace, fill volume: 50 mL flasks containing approx. 50 mL of test medium
- Aeration: Continuously stirred
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 5000 cells per mL
- Control end cells density: mean; 41.047 x 10^4 mL; standard deviation; 8.338 x 10^4 mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD medium) prepared according to guideline.
- Total organic carbon: < LOQ
- Particulate matter: Not reported
- Conductivity: < 5 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH measured in all test concentrations and the control at the start and end of the test. Temperature measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: None
- Photoperiod: Continuous
- Light intensity and quality: Mean: 6720 Lux (range: 6250 – 7100 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth rate and yield at 72 hours
- Determination of cell concentrations: Determined by spectrophotometer at 24, 48 and 72 hours
- Chlorophyll measurement: None

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None; Limit test performed
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: Range finding study conducted but the concentrations are not reported. The study states concentrations above the limit concentration (100 mg/L or limit of solubility) were not tested in accordance with guidelines.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95 % confidence interval could not be determined
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities observed microscopically
- Unusual cell shape: None reported
- Colour differences: None
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Other: The validity criteria were met as:
Increase in cell density by at least a factor of 16 within 72 hours in the control cultures (observed; 82.1-fold increase)
The coefficient of variation of sectional (daily) growth rates in control cultures must not exceed 35 % (observed; 24 %)
The coefficient of variation of average growth rates between control replicates must not exceed 7 % (observed; 5 %)
- Any stimulation of growth found in any treatment: Stimulation observed in the 100 mg/L test concentration, the only concentration tested
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: No effects observed
Results with reference substance (positive control):
Reference substance results not reported
Reported statistics and error estimates:
The test item growth and yield values were compared to the control with a Student t-test (α = 0.05, one-sided). The NOEC and LOEC were calculated using appropriate statistical methods

Table 1: Influenece of the test item on the Growth Pseudokirchneriella subcapitata

 Parameter (0 - 72 hours)  Growth rate (mg test item/L)  Yield (mg test item/L)
 72 -hour EC50  > 100 (nominal)  > 100 (nominal)
 95 % conf. limits  n.d.  n.d.
 72 -hour EC10  > 100 (nominal)  > 100 (nominal)
 95 % conf. limits  n.d.  n.d.
 72 -hour NOEC   100 (nominal)   100 (nominal)
 72 -hour LOEC  > 100 (nominal)  > 100 (nominal)

n.d. not determinable

Table 2: Algal Cell Densities During the Test Period of 72 Hours

Nominal Concentration (mg test item/L)

 Flask No.

 Density of algal cells (10^4/mL) after

 24 hours

 48 hours

 72 hours

 Control

 1

 1.384

 10.128

 49.948

 2  1.519  7.841  48.199
 3  1.653  7.303  41.742
 4  1.519  6.362  34.478
 5  1.519  5.285  28.155
 6  1.384  7.438  43.760
 m  1.496  7.393  41.047
 s  0.101  1.625  8.338
 100  1  1.922  7.572  53.850
 2  1.788  4.344  44.836
 3  1.519  4.882  40.531
 4  1.250  9.187  52.773
 5  1.250  12.012  57.347
 6  1.519  9.187  38.783
 m  1.541  7.864  48.020
 s  0.275  2.901  7.683

m: mean value; s: standard deviation

At test start 5000 algal cells/mL were incubated

Table 3: Growth Rates µ and Percentage Inhibition of µ During the Test Period

Nominal Concentration (mg/L)  Growth rates µ (1/day) and % inhibition of µ    
 0 - 24 hours  0 - 48 hours  0 - 72 hours
 µ  %    µ  %    µ  %  
 Control

 1.094

 0.0

 

 1.337

 0.0

 

 1.463

 0.0

 

 100

 1.112

 -1.7

 -

 1.347

 -0.7

 -

 1.518

 -3.8

 -

negative values in '% inhibition' indicate an increase in growth relative to that of the control

- no significant difference compared to the control (tested with Student t-test, α = 0.05, one-sided)

Table 4: Yield, y, and Percentage Inhibition of y During the Test Period

Nominal Concentration (mg/L)

 Yield y (x*10^4 cells/mL) and % inhibition of y    
 0 - 24 hours  0 - 48 hours  0 - 72 hours
 y  %    y  %    y  %  
 Control  0.996  0.0    6.893  0.0    40.547  0.0  
 100  1.041  -4.5  -  7.364  -6.8  -  47.250  -17.2  -

negative values in '% inhibition' indicate an increase in growth relative to that of the control

- no significant difference compared to the control (tested with Student t-test,α= 0.05, one-sided)

Table 5: Appearance of the Test Item in Test medium

Nominal Concetration (mg/L)    

Appearance of the test item in the test medium

Start (0 hours)

 24 hours

 48 hours

 End (72 hours)

 100

 0

 0

 0

 0

0 - No remarkable observations, clear test medium

Table 6: pH-Values in the Test Media at the Start and the End of the Test

Nominal Concentration

 pH value

 (mg test item/L)

 Start (0 hours)

 End (72 hours)

 Control

 8.1

 7.9

 100

 8.1

 8.0

Table 7: Temperature in the Test Medium During the Test Period

    

Water Temperature (°C)

Start (0 hours)

 24 hours

 48 hours

 End (72 hours)

 Control

 23

 23

 23

 23

Table 8: Results for the Determination of the Test Item in the Test Samples

 Sample Description

 Total Carbon

 Inorganic Carbon 

 Total Organic Carbon (TOC)

 mg/L

 age (h)

 Found (mg/L)

 DF

 Calculated (mg Carbon/L)1

 Found (mg/l)

 DF

 Calculated (mg/Carbon/l)1

 (mg Carbon/L)

 Control

 0

 4.94

 1

 4.94

 5.72

 1

 5.72

 <LOQ

 Control

 0

 4.82

 1

 4.82

 6.53

 1

 6.53

  <LOQ

100

 0

 4.73

 1

 4.73

 5.50

 1

 5.50

  <LOQ

100

 0

 4.62

 1

 4.62

 5.47

 1

 5.47

  <LOQ

 100

 72

 4.61

 1

 4.61

 6.10

 1

 6.10

  <LOQ

 100

 72

 4.55

 1

 4.55

 6.21

 1

 6.21

  <LOQ

1The tabulated results represent rounded results calculated on the exact raw data

LOQ: Limit of Quantification = 3 mg Carbon/L

D.F. Dilution Factor

Validity criteria fulfilled:
yes
Remarks:
Increase in cell density observed: 82.1-fold increase. The coefficient of variation of sectional growth rates in control observed: 24 %. Coefficient of variation of average growth rates between control replicates observed: 5 %
Conclusions:
The toxicity of the test item to the green algae Pseudokirchneriella subcapitata in 72-hour limit test was investigated. Based on nominal concentrations the EC10 and EC50 for growth rate and yield were determined to be >100 mg/L. The NOEC and LOEC were determined to be ≥100 and >100 mg/L, respectively.
Executive summary:

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was investigated in 72-hour limit test was according to the OECD 201 guideline. The study was conducted as a limit test using a water accommodated fraction due to the test item being a complex UVCB. The test item was prepared as a water accommodated fraction by adding 200 mg of the test item to 2000 mL of test water and slightly stirring for 24 hours; the aqueous phase was then drawn off and used in the test. A control, containing test water only, was conducted in parallel. Six replicates of the control and test item were performed. Analysis of the test item concentrations was performed by calibrated TOC analysis at test start and test end. The cell density all solutions was determined at 24, 48 and 72 hours, and percentage inhibition of growth rate and yield was determined at 72 hours.

Based on nominal concentrations the EC10 and EC50 for growth rate and yield were determined to be >100 mg/L, the only concentration tested. The NOEC and LOEC were determined to be ≥100 and >100 mg/L, respectively.

The study is a GLP compliant guideline experimental study fully acceptable for assessment of this endpoint.

Description of key information

Toxicity to aquatic algae has been assessed for three substances that would fall within this TDI-I category definition. All studies used water accommodated fractions as the test items are poorly soluble UVCBs. All but one of the studies determined EL50 values of >100 mg/L, with NOELR values of 100 mg/L.

One study conducted with Diurea 8 in base grease did show effects at <100 mg/L, but this was considered to be due to impurities in the sample, and therefore the study was repeated on both the isolated substance and the substance in base grease. Both of these studies showed EL50 values >100 mg/L WAF and NOELR values of 100 mg/L WAF. The first study is therefore disregarded due to impurities in the test sample. 

Based on these results, members of the TDI-I category are not expected to be toxic to aquatic algae at the limit of solubility.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Five studies were conducted on three different TDI-I category members in order to assess toxicity to aquatic algae. Studies were conducted with Tetraurea 2 in base grease and Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine in the isolated form. Three separate studies were conducted with Diurea 8; two studies were conducted on Diurea 8 in base grease and one on the isolated Diurea 8 thickener. The results of these studies are discussed below.

Diurea 8

Vryenhoef (2011b)

A study was performed to assess the effect of Diurea 8 (in base grease) on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD guideline 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of Diurea 8 over a range of nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

The effect of Diurea 8 (in base grease) on the growth of Pseudokirchneriella subcapitata over a 72-Hour period gave a growth rate EL50 of 52 mg/L (95% Confidence Limits: 50 - 53 mg/L) and a yield EL50 of 36 mg/L (95% Confidence Limits: 32 – 39 mg/L). For both growth rate and yield, the No Observed Effect Loading Rate (NOELR) was 25 mg/L and the Lowest Observed Effect Loading Rate (LOELR) was 50 mg/L.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given that measured concentrations of less than the limit of quantification (LOQ), which was considered to be 1.0 mg C/L, were obtained for the control and all test loading rates, it was considered that the results gave no evidence of the presence of Diurea 8 in the WAF.

Vryenhoef (2012a)

A study was performed to assess the effect of the isolated Diurea 8 thickener on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD guideline 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of Diurea 8, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

The effect of Diurea 8 (isolated thickener) on the growth of Pseudokirchneriella subcapitata gave EL50 (effective loading rate) values of greater than 100 mg/L WAF. Correspondingly the No Observed Effect Loading Rate was 100 mg/L WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

 

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured concentrations of less than the limit of quantification (LOQ) were obtained which was considered to be 1.0 mg C/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to Diurea 8 as a whole the results were based on nominal loading rates only.

Vryenhoef (2012b)

The study was performed to assess the effect of the test item (in base grease) on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD guideline 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of Diurea 8, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

The effect of Diurea 8 (in base grease) on the growth ofPseudokirchneriella subcapitatagave EL50 (effective loading rate) values of greater than 100 mg/L WAF. Correspondingly the No Observed Effect Loading Rate (NOELR) was 100 mg/L WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured test concentrations to be below or around the limit of quantification (LOQ) which was considered to be 1.0 mg C/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test items as a whole, and the dissolved concentrations were at or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Tetraurea 2

Vryenhoef (2011a)

The study was performed to assess the effect of the test item (in base grease) on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD guideline 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of Tetraurea 2, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

The effect of Tetraurea 2 on the growth of Pseudokirchneriella subcapitata gave EL50 (effective loading rate) values of greater than 100 mg/L WAF. Correspondingly the No Observed Effect Loading Rate (NOELR) was 100 mg/L WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours and showed measured test concentrations to be below or around the limit of quantification (LOQ) which was considered to be 1.0 mg C/L. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test items as a whole, and the dissolved concentrations were at or below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine

Kley and Wydra (2010b)

The toxicity of the test item to the green algae Pseudokirchneriella subcapitata was investigated in GLP compliant 72 hour limit test according to the OECD 201 guideline. The study was conducted as a limit test using a water accommodated fraction due to the test item being a complex UVCB. The test solutions were prepared by adding 200 mg of the test item to 2000 mL of test water and slightly stirring for 24 hours; the aqueous phase was then drawn off and used in the test. Analysis of the test item concentrations was performed by calibrated TOC analysis at the test start and test end. The cell density of all solutions was determined at 24, 48 and 72 hours, and percentage inhibition of growth rate and yield was determined at 72 hours.

Based on nominal concentrations the EC10 and EC50 for growth rate and yield were determined to be > 100 mg/L, the only concentration tested. The NOEC was determined to be ≥ 100 mg/L.

 Summary of available algae toxicity data for TDI-I category members

The level of toxicity observed in the study Vryenhoef (2011b) was contrary to what was expected for a clean base grease (i.e. simply the base oil plus Diurea 8 thickener prior to the addition of any other performance additives). There were concerns that the base grease sample supplied for this study may have contained additional performance additives. To address these concerns a fresh batch of Diurea 8 base grease was prepared and a second acute algae toxicity study was conducted (Vryenhoef 2012b). The second study was conducted as a limit test at 100 mg/L WAF, based on the results of a preliminary range finding study. The results gave EL50 (effective loading rate) values of greater than 100 mg/L WAF for both growth rate and yield. Correspondingly, the No Observed Effect Loading Rate (NOELR) was 100 mg/L WAF. A study was also conducted with the isolated Diurea 8 thickener (Vryenhoef 2012a) and also showed EL50 values of >100 mg/L WAF and a NOELR of 100 mg/L WAF.

The results of the repeated base-grease studies were in agreement with the results of the other studies conducted on TDI-category I substances (in both the grease base and isolated forms) and therefore it can be concluded that the second test (Vryenhoef 2012b) with the base grease containing the Diurea 8 thickener presents a more relevant and reliable result for the toxicity to algae than the first study (Vyrenhoef 2011b).

In summary, data are available which show that both the isolated TDI-I category members and TDI-I category members in base grease are not toxic to algae at nominal loading rates of up to 100 mg/L. These results are comparable to the observed toxicity of category members to other trophic levels and reflect the lack of toxicity expected with very low water solubility and limited bioavailability in the grease matrix.