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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 March 2010 and 29 April 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In water aluminium orthophosphates will dissociate into their ionic forms (Al3+ and PO43-, which will further associate with the ionic forms of H2O). It is therefore considered acceptable to assess the aluminium and phosphate ions as separate entities.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
No additional information.
3. ANALOGUE APPROACH JUSTIFICATION
This study is submitted as part of a weight of evidence approach in accordance with REACH Annex XI, Section 1.2. When considered alongside the additional data provided for this endpoint it is considered that the ecotoxicity of the substance to be registered is adequately assessed.
4. DATA MATRIX
See full read-across justification attached under Section 13 of this dossier and summary of data included in the endpoint summary under Section 6.1 of this dossier.
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15/09/2009 Date of Signature on GLP certificate: 26/11/2009
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Chemical name: Phosphoric acid, potassium salt (2:3), dihydrate.
Synonyms (IUPAC name): Tripotassium trihydrogen diphosphate dihydrate.
Chemical formula: H3 O4 P . H2 O . 3/2 K
CAS number: 6922-99-4
Molecular weight: 346.29
Melting range: No complete melting up to 573 K
Solubility in water ( g/L, at 20 °C): 705 (pH 7)
Analytical monitoring:
yes
Details on sampling:
See Appendix 1
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test item was dissolved directly in culture medium.
An amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution. This stock solution was inoculated with algal suspension (6.9 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.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: Strain CCAP 276/20
- Source: Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, 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 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).
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 deg C until the algal cell density was approximately 104 - 105 cells/ml.

ACCLIMATION
- Acclimation period: Not recorded.

- 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.

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* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


- Any deformed or abnormal cells observed: None recorded.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.0 pH meter.
Dissolved oxygen:
Not recorded
Salinity:
Not applicable
Nominal and measured concentrations:
The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 1.0, 10 and 100 mg/l for a period of 72 hours.
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.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 107% to 113% of nominal and so the results are based on nominal test concentrations only.
Details on test conditions:
EXAMPLE
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 5.76 x 105 cells per ml. Inoculation of 1 litre of test medium with 6.9 ml of this algal suspension gave an initial nominal cell density of 4 x 103 cells per ml and had no significant dilution effect on the final test concentration.

Mean cell density of control at 0 hours : 4.12 x 103 cells per ml
Mean cell density of control at 72 hours : 4.02 x 105 cells per ml


- No. of organisms per vessel:
initial cell density of approximately 103 cells/ml

- No. of vessels per concentration (replicates):
Six replicate flasks per concentration (in definitive test).

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


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

TEST MEDIUM

An 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 and 1.0 mg/l. An aliquot (200 ml) of each of the stock solutions was separately inoculated with algal suspension (4.7 ml) to give the required test concentrations of 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.
The control group was maintained under identical conditions but not exposed to the test item.

OTHER TEST CONDITIONS

- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by 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 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 5.76 x 105 cells per ml. Inoculation of 1 litre of test medium with 6.9 ml of this algal suspension gave an initial nominal cell density of 4 x 103 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 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.

- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
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.

- Range finding study:
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 Desmodesmus subspicatus cells to a series of nominal test concentrations of 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 material.

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

POSITIVE CONTROL:
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.


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:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
phosphate
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
phosphate
Basis for effect:
growth rate
Details on results:
Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test item during the range-finding test are given in Table 1.
The results showed no effect on growth at the test concentrations of 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.

Definitive Test

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.


Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 98 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 : 4.12 x 103 cells per ml
Mean cell density of control at 72 hours : 4.02 x 105 cells per ml
The mean coefficient of variation for section by section specific growth rate for the control cultures was 28% 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 Desmodesmus subspicatus (CCAP 276/20) were not affected by the presence of the test item at a nominal test concentration of 100 mg/l over the 72-Hour exposure period.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.
Accordingly the following results were determined from the data:
5.2.2.1 Inhibition of growth rate
ErC10 (0 - 72 h) : >100 mg/l
ErC20 (0 - 72 h) : >100 mg/l
ErC50 (0 - 72 h) : >100 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 100 mg/l 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 test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/l.

Inhibition of yield

EyC10 (0 - 72 h) : >100 mg/l
EyC20 (0 - 72 h) : >100 mg/l
EyC50 (0 - 72 h) : >100 mg/l
where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as in Section 5.2.2.1. There were no statistically significant differences (P0.05), between the control and 100 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/l.

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.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 0039/1127) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference item gave the following results:
ErC50 (0 – 72 h) : 0.49 mg/l*
EyC50 (0 – 72 h) : 0.18 mg/l, 95% confidence limits 0.16 – 0.21 mg/l
No Observed Effect Concentration (NOEC) based on growth rate: 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/l
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Table 1. 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

4.26E+03

2.58E+05

 

 

 

R2

4.14E+03

2.50E+05

-

-

 

Mean

4.20E+03

2.54E+05

 

 

1.0

R1

3.89E+03

2.80E+05

 

 

 

R2

4.17E+03

2.80E+05

[4]

[10]

 

Mean

4.03E+03

2.80E+05

 

 

10

R1

4.10E+03

2.63E+05

 

 

 

R2

4.66E+03

2.32E+05

2

3

 

Mean

4.38E+03

2.48E+05

 

 

100

R1

4.69E+03

2.50E+05

 

 

 

R2

4.03E+03

2.78E+05

0

[4]

 

Mean

4.36E+03

2.64E+05

 

 

[ ]


Table 2. Cell Densities and pH Values in the DefinitiveTest

Nominal Concentration

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.2

4.66E+03

1.43E+04

8.74E+04

4.80E+05

7.8

 

R2

7.2

3.61E+03

1.20E+04

8.50E+04

3.64E+05

7.8

 

R3

7.2

3.70E+03

1.31E+04

8.12E+04

2.85E+05

7.8

 

R4

7.2

3.94E+03

1.14E+04

1.09E+05

4.65E+05

7.8

 

R5

7.2

4.54E+03

1.36E+04

9.36E+04

3.45E+05

7.8

 

R6

7.2

4.24E+03

1.07E+04

8.44E+04

4.76E+05

7.8

 

Mean

 

4.12E+03

1.25E+04

9.01E+04

4.02E+05

 

100

R1

7.0

4.22E+03

1.90E+04

1.06E+05

4.55E+05

7.5

 

R2

7.0

3.57E+03

1.79E+04

7.50E+04

3.25E+05

7.5

 

R3

7.0

4.10E+03

1.62E+04

7.70E+04

2.84E+05

7.5

 

R4

7.0

4.00E+03

1.73E+04

1.05E+05

3.94E+05

7.5

 

R5

7.0

4.06E+03

1.49E+04

1.08E+05

4.03E+05

7.5

 

R6

7.0

4.92E+03

1.22E+04

1.09E+05

4.45E+05

7.5

 

Mean

 

4.15E+03

1.62E+04

9.67E+04

3.84E+05

 

 


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

0.053

0.075

0.071

 

R2

0.046

0.082

0.061

 

R3

0.050

0.076

0.052

 

R4

0.044

0.094

0.061

 

R5

0.051

0.080

0.054

 

R6

0.041

0.086

0.072

 

Mean

0.048

0.082

0.062

 

Table 4.  Inhibition of Growth Rate and Yieldin the Definitive Test

Nominal Concentration
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.066

 

4.75E+05

 

 

R2

0.063

 

3.61E+05

 

 

R3

0.059

 

2.81E+05

 

 

R4

0.066

-

4.61E+05

-

 

R5

0.062

 

3.40E+05

 

 

R6

0.066

 

4.71E+05

 

 

Mean

0.064

 

3.98E+05

 

 

SD

0.003

 

8.21E+04

 

100

R1

0.066

[3]

4.51E+05

 

 

R2

0.061

5

3.21E+05

 

 

R3

0.059

8

2.79E+05

 

 

R4

0.064

0

3.90E+05

 

 

R5

0.064

0

3.99E+05

 

 

R6

0.065

[2]

4.40E+05

 

 

Mean

0.063

1

3.80E+05

5

 

SD

0.003

 

6.75E+04

 

[ ]


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

R1- R6= Replicates 1 to 6

R1- R6= Replicates 1 to 6

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

R1– R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Read-across is justified on the basis detailed in rationale for reliability above. This study is therefore considered to be of sufficient adequacy and reliability to be used as a key study and no further testing is justified.

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

In accordance with Annex VII, section 9.1.2, column 2 of Regulation (EC) No. 1907/2006 (REACH) a growth inhibition study in aquatic plants does not need to be conducted if there are mitigating circumstances. As a result of a technical assessment of the feasibility of conducting an algal inhibition study on aluminium orthophosphate (EC No. 232-056-9), conducted at Harlan Laboratories Ltd, no study for algal growth inhibition has been performed. 

 

The purpose of this investigation was to determine the solubility of the test material in algal culture medium and to assess the effect of the dissolved test material on the pH. As the effects of a reduction in the pH, due to increasing concentrations, is considered to pose a risk of toxicity due to pH (as opposed to the intrinsic toxicity of the test material). If the test material is considered to have a significant effect on the pH, pH adjustment would be required and this may result in precipitation of Al which would in turn invalidate the study.

 

Aluminium orthophosphate has been selected for investigation on the basis that this substance contains the greatest amount of aluminium (%w/w) in comparison to the following aluminium phosphates:

-         aluminium tris(dihydrogen phosphate) (EC No. 236-875-2)

-         sodium aluminium phosphate (EC No. 232-090-4)

Therefore the data can be used for read-across to assess the toxicological impact of aluminium as phosphate is not considered to be toxic to algae.

The results of the preliminary work on aluminium orthophosphate are as follows:

An initial test item dispersion of 1000 mg/l was prepared by dispersing 500 mg of test item in 500 ml of algal culture medium in a ground glass stoppered conical flask.  The test item dispersion was shaken at 300 rpm at a temperature of 30oC for 24 hours.  After the shaking period, the test item dispersion was allowed to stand for 24 hours at 21oC prior to removal of the undissolved test item by centrifugation (13500 rpm, 15 minutes).

 

The resultant solution had a pH of 6.95.  The pH of this solution was adjusted using 1M NaOH to pH 7.5 after which fine particles of precipitate were observed to be dispersed throughout the test medium.

 

Coulter®counts performed on the solution pre- and post-pH adjustment showed an increase in particles within the 2-20 mm range after pH adjustment.

 

The results suggest that whilst it may be technically possible to conduct the algal growth inhibition study in accordance with OECD guideline 201 after centrifugation of the post-pH adjusted ‘solution’ to remove any particulates, this would mean that testing would be conducted at concentrations below the water solubility of the test item and therefore the results of such a study would be subject to doubt. In addition further precipitation may occur during the test as a result of a rise in pH due to algal growth which would lower the photosynthesis rates due to light reflection and thus influence growth rates due to physical reasons and not for toxicological reasons.

In addition to assess the toxicity of phosphate a key study on an analogous phosphate substance is also submitted.

Based on the available read across data aluminium orthophosphate is considered not acutely toxic to aquatic algae.