Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 06 December 2016 and 04 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Identification: Diammonium phthalate
Physical state/Appearance: White crystalline powder
Batch: 7172002
Purity: 99.3%
Expiry Date: 01 August 2017
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-finding test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive test
Samples were taken from the control and the 100 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Range-finding test
A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.7 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive test
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution. This stock solution was inoculated with algal suspension (8.2 mL) to give the required test concentration of 100 mg/L.
The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

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 ± 2 °C.

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 ± 1 °C until the algal cell density was approximately 10E4 – 10E5 cells/mL.

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
24 ± 1 ºC
pH:
7.7 - 8.0
Nominal and measured concentrations:
Nominal concentrations
Definitive test 100 mg/L

Measured concentrations
The geometric mean measured test concentration was determined to be 22 mg/L.

See results section for verification of test concentrations for further details.
Details on test conditions:
Range-Finding Test
The test concentration 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 test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.
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 ± 1 ºC 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.

Definitive Test
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines, no effect on algal growth was observed.

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 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 characterized by a cell density of 6.09 x 10^5 cells per mL. Inoculation of 1 liter of test medium with 8.2 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 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.

Assessments
Test Organism Observations
Samples were taken at 26, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria
The pH of the control and 100 mg/L test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: inhibition of yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: inhibition of yield
Details on results:
Range-finding Test:
The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines, no effect on growth was observed.
Chemical analysis of the 100 mg/L test preparations at 0 and 72 hours showed near nominal concentrations were obtained.

Definitive Test:
Verification of Test Concentrations:
Analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 96% of nominal was obtained. A decline in measured concentration was observed at 72 hours to 5% of nominal.
This effect contradicted the results obtained from the analysis of the range-finding test preparations where there was no significant decline in measured concentration over the 72-Hour test period. However, a decline in measured concentrations was observed in the Acute Toxicity to Daphnia magna range-finding test (Study Number FX47YT) suggesting that the test item was unstable. It is believed that there may have been a mix up with the sampling during analysis of the range-finding test samples and that the 0-Hour 100 mg/L test sample may have been analyzed twice.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 22 mg/L.

Growth Data:
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 nominal concentrations in excess of 100 mg/L.
Accordingly the following results were determined from the data based on the geometric mean measured test concentration:

Inhibition of growth rate
ErC10 (0 - 72 h) : >22 mg/L
ErC20 (0 - 72 h) : >22 mg/L
ErC50 (0 - 72 h) : >22 mg/L

where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and 22 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There was no statistically significant decrease in growth rate (P ≥ 0.05), between the control and 22 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 22 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : >22 mg/L
EyC20 (0 - 72 h) : >22 mg/L
EyC50 (0 - 72 h) : >22 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There was no statistically significant decrease in yield (P ≥ 0.05), between the control and 22 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 22 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 106 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 : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 5.31 x 10^5 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%.

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 cultures, however unidentified micro-organisms were observed to be present in the 100 mg/L test cultures.



Results with reference substance (positive control):
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.

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 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 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.

Any other information on results incl. tables

Water Quality Criteria

Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.0 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.

Observations on Test Item Solubility

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

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

Nominal Concentration

(mg/L)

Cell Densities* (cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

6.38E+03

8.41E+05

-

-

R2

6.48E+03

1.00E+06

Mean

6.43E+03

9.21E+05

0.10

R1

6.58E+03

9.73E+05

0

0

R2

6.58E+03

8.73E+05

Mean

6.58E+03

9.23E+05

1.0

R1

7.68E+03

1.37E+06

[3]

[37]

R2

7.60E+03

1.16E+06

Mean

7.64E+03

1.26E+06

10

R1

6.40E+03

1.09E+06

[3]

[16]

R2

6.74E+03

1.04E+06

Mean

6.57E+03

1.07E+06

100

R1

6.21E+03

1.19E+06

[7]

[46]

R2

6.83E+03

1.50E+06

Mean

6.52E+03

1.35E+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]

Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration

(mg/L)

pH

Cell Densities*(cells per mL)

pH

0 h

26 h

48 h

72 h

72 h

Control

R1

7.7

2.45E+04

9.68E+04

4.72E+05

8.0

R2

2.32E+04

9.28E+04

4.36E+05

R3

2.47E+04

9.57E+04

4.97E+05

R4

2.48E+04

1.09E+05

6.28E+05

R5

2.51E+04

9.96E+04

5.56E+05

R6

2.57E+04

1.08E+05

5.99E+05

Mean

2.47E+04

1.00E+05

5.31E+05

22

R1

7.5

2.28E+04

9.69E+04

5.95E+05

8.0

R2

2.45E+04

9.81E+04

6.00E+05

R3

2.46E+04

1.06E+05

7.36E+05

R4

2.27E+04

8.84E+04

6.49E+05

R5

2.49E+04

1.00E+05

6.90E+05

R6

2.30E+04

1.01E+05

7.26E+05

Mean

2.38E+04

9.86E+04

6.66E+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

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

0.061

0.062

0.066

R2

0.059

0.063

0.064

R3

0.061

0.062

0.069

R4

0.062

0.067

0.073

R5

0.062

0.063

0.072

R6

0.063

0.065

0.071

Mean

0.061

0.064

0.069

 


R1- R6= Replicates 1 to 6

Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.063

 

4.67E+05

 

R2

0.062

 

4.31E+05

 

R3

0.064

 

4.92E+05

 

R4

0.067

-

6.23E+05

-

R5

0.065

 

5.51E+05

 

R6

0.066

 

5.94E+05

 

Mean

0.065

 

5.26E+05

 

SD

0.002

 

7.52E+04

 

22

R1

0.066

[2]

5.90E+05

 

R2

0.066

[2]

5.95E+05

 

R3

0.069

[6]

7.31E+05

 

R4

0.068

[5]

6.44E+05

 

R5

0.068

[5]

6.85E+05

 

R6

0.069

[6]

7.21E+05

 

Mean

0.068

[4]

6.61E+05

[26]

SD

0.001

 

6.13E+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]

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on the geometric mean measured test concentration gave EC50 values of greater than 22 mg/L. The No Observed Effect Concentration was 22 mg/L.
Executive summary:

 Introduction

A study was perford 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) No 761/2009.

Methods.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a nominal concentration 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.

Analysis of the 100 mg/L test preparation at 0 hours showed a measured test concentration of 96% of nominal was obtained. A decline in measured concentration was observed at 72 hours to 5% of nominal.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the geometric mean measured test concentration of greater than 22 mg/L. The No Observed Effect Concentration was 22 mg/L.

It was considered unnecessary and unrealistic to test at nominal concentrations in excess of 100 mg/L.