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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:
31 March 2014 to 2 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken at 0, 24, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

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.

Verification of Test Concentrations:

Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All 0-Hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

In order to determine whether the decline in measured test concentration observed in the range­ finding test was due to instability and/or adsorption of the test item to the algal cells present, an additional sample of the 100% v/v saturated solution test concentration containing no algal cells
was prepared at the start of the test. This sample was incubated alongside the test prior to analysis at 72 hours.
Vehicle:
no
Details on test solutions:
Range-finding test:

The results obtained from the preliminary media preparation trial conducted indicated that a saturated solution method of preparation followed by the removal of any undissolved test item by centrifugation was most appropriate for this test item.

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 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

An amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10 and 1.0% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.3 mL) to give the required test concentrations of 1.0, 10 and 100% v/v saturated solution.

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 used for each control and test concentration.

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 °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.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

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. Only concentrations within the range to be used for the definitive test were analyzed.

Definitive Test:

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no effect on algal growth was observed.

Experimental Preparation:

An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100% v/v saturated solution. An aliquot (1 liter) of each of the saturated solution was inoculated with 4.8 mL of algal suspension to give the required test concentration of 100% v/v saturated solution.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.



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, Ohan, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °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 104 - 105 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not measured
Test temperature:
24 ± 1 °C
pH:
The pH value of the control was 7.9 when measured at 0 and 72 hours.
The pH value of the 100% v/v saturated solution was 8.0 when measured at 0 and 72 hours.
Dissolved oxygen:
Not measured
Salinity:
Not applicable
Nominal and measured concentrations:
Analysis of the 100% v/v saturated solution test preparation at 0 hours showed a measured lanthanum concentration of 0.0015 mg/L; equivalent to a test item concentration of 0.0022 mg/L was obtained. A decline in the measured lanthanum concentration was observed at 72 hours to 0.0011 mg/L; equivalent to a test item concentration of 0.0015 mg/L. Analysis of an additional sample at 72 hours prepared with the omission of algal cells showed a measured lanthanum concentration of 0.0032 mg/L; equivalent to a test item concentration of 0.0045 mg/L was obtained.
Details on test conditions:
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 104 - 105 cells/mL.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
biomass
Details on results:
Validation criteria:

The following data show that the cell concentration of the control cultures increased by a factor of 152 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 Mean cell density of control at 72 hours

5.18 x 103 cells per mL
7.88 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 29% 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. There were no abnormalities detected in any of the control or test cultures.

Water Quality Criteria:

The pH value of the control was 7.9 when measured at 0 and 72 hours.
The pH value of the 100% v/v saturated solution was 8.0 when measured at 0 and 72 hours.
Temperature was maintained at 24 ± 1 °C throughout the test.

The pH value of the control cultures (see Table 2) was determined to be pH 7.9 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.

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 green dispersions.

CONCLUSION

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 0.0022 mg/L based on the 0-Hour measured test concentrations. The No Observed Effect Concentration based on inhibition of growth rate and yield was 0.0022 mg/L.

This study showed that there were no toxic effects at saturation.


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) 1.1 mg/L; 95% confidence limits 0.91 - 1.2 mg/L
EyC50 (0 - 72 h) 0.51 mg/L; 95% confidence limits 0.45 - 0.59 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:
Statistical analysis of the yield data was carried out.There were no statistically significant differences (P0.05), between the control and 0.0022 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.0022 mg/L.

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

 

 

Nominal Concentration

(%v/vSaturatedSolution)

CellDensities*(cellspermL)

InhibitionValues(%)

 

0Hours

 

72Hours

 

GrowthRate

 

Yield

 

 

Control

R1

5.42E+03

6.42E+05

 

-

 

-

Rz

5.38E+03

6.29E+05

Mean

5.40E+03

6.35E+05

 

 

1.0

R1

5.44E+03

7.16E+05

 

 

[5]

 

 

[22]

Rz

5.41E+03

8.28E+05

Mean

5.42E+03

7.72E+05

 

 

10

R1

5.76E+03

6.77E+05

 

 

2

 

 

0

Rz

5.79E+03

5.97E+05

Mean

5.78E+03

6.37E+05

 

 

100

R1

6.37E+03

6.38E+05

 

 

0

 

 

[1]

Rz

4.70E+03

6.43E+05

Mean

5.54E+03

6.41E+05

 

 Inhibition of Growth Rate and Yield in the Definitive Test

 

Nominal Concentration

(%v/vSaturatedSolution)

GrowthRate(cells/mL/hour)

Yield(cells/mL)

0-72h

%Inhibition

0-72h

%Inhibition*

 

 

 

 

 

Control

R1

0.072

 

8.69E+05

 

Rz

0.069

 

7.l8E+05

 

RJ

0.068

 

6.82E+05

 

Ri

0.070

-

7.77E+05

-

Rs

0.071

 

7.96E+05

 

 

0.072

 

8.56E+05

 

Mean

0.070

 

7.83E+05

 

SD

0.002

 

7.41E+04

 

 

 

 

 

 

100

R1

0.069

l

6.96E+05

 

Rz

0.069

1

7.37E+05

 

RJ

0.069

1

7.39E+05

 

Ri

0.071

[l]

8.01E+05

 

Rs

0.068

3

6.67E+05

 

 

0.068

3

6.85E+05

 

Mean

0.069

1

7.21E+05

8

SD

0.001

 

4.87E+04

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 0.0022 mg/L. The No Observed Effect Concentration based on inhibition of growth rate and yield was determined to be 0.0022 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 0.0022 mg/L. The No Observed Effect Concentration based on inhibition of growth rate and yield was determined to be 0.0022 mg/L.

This study data has been read-across from the similar substance lanthanum trifluoride.

This study showed that there were no toxic effects at saturation.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across to structurally similar substance, lanthanum trifluoride, is justified on the basis that ecotoxicological effects will be driven by the metal cation species (La3+) which is analogous to the registered substance and the read-across substance.
Reason / purpose for cross-reference:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.002 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Basis for effect:
growth rate

Description of key information

No effects on toxicity to aquatic algae up to the limit of water solubility. Pseudokirchneriella subcapitata, OECD 201, Harlan 2014 (read across from lanthanum trifluoride).

Key value for chemical safety assessment

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria of Klimisch (1997).

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 0.0022 mg/L. The No Observed Effect Concentration based on inhibition of growth rate and yield was determined to be 0.0022 mg/L. In summary,

no effects on toxicity to aquatic algae up to the limit of water solubility

No algal toxicity studies are available for the registered substance, lanthanum trihydroxide. A valid study has been provided on the structural analogue, lanthanum trifluoride. Read-across to this structurally similar substance is justified on the basis that ecotoxicological effects will be driven by the metal cation species (La3+) which is analogous to the registered substance and the read-across substance.