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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:
The study was conducted between1 May 2015 and 20 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations
Samples were taken from the control and each 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.
Vehicle:
no
Details on test solutions:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.
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 3.3). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
7.6 - 8.5
Salinity:
Not applicable, as freshwater study
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal concentrations: 0.1, 1.0, 10 and 100 mg/L

DEFINITIVE TES
- Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Measured concentrations at t=0h: 0.867, 2.87, 9.01, 29.7 and 94.9 mg/L
- Measured concentrations at t=72h: 0.67, 2.17, 7.34, 24.9 and 84.9 mg/L
- Measured concentrations geomean t=0h and t=72h: 0.76, 2.5, 8.1, 27 and 90 mg/L
Details on test conditions:
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 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 characterized by a cell density of 5.31 x 105 cells per mL. Inoculation of 500 mL of test medium with 4.7 mL of this algal suspension gave an initial nominal cell density of 5 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L.: 7.4 - 12 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
8.1 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.:
3.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% C.L.: 2.9 - 4.2 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Range-finding Test
The results showed no effect on growth rate at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Growth Data
From the data given,, 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
ErC10 (0 - 72 h) : 2.9 mg/L
ErC20 (0 - 72 h) : 4.5 mg/L
ErC50 (0 - 72 h) : 9.5 mg/L; 95% confidence limits 7.4 - 12 mg/L

where ErCx is the test concentration that reduced growth rate by x%.
Results with reference substance (positive control):
Positive Control
A positive control (Harlan Study Number 41403074) 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) : 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/L
EyC50 (0 – 72 h) : 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 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 growth rate data was carried out for the control and all test concentrations 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 (P³0.05), between the control, 0.76 and 2.5 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 2.5 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 8.1 mg/L.

Table: Inhibition of griowth rate and yield

Nominal conc. (mg/L)

 

growth rate

Yield

0-72 h

% inhibition

0-72 h

% inhibition

control

0.075

-

1.10E+06

-

1.0

0.074

1

1.05E+06

4

3.2

0.070

7

7.65E+05

30

10

0.043

73

1.11E+05

90

32

0.009

88

3.94E+03

100

100

0.002

98

-2.03E+02

100

Inhibition of Yield

EyC10(0 - 72 h)   :  1.5mg/L

EyC20(0 - 72 h)   :   2.0 mg/L

EyC50(0 - 72 h)   :   3.5 mg/L; 95% confidence limits 2.9 – 4.2 mg/L

 

Where:

 

EyCxis the test concentration that reduced yield by x%.

 

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences (P³0.05), between the control and 0.76 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.76 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 2.5 mg/L.

 

 Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 205 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                      :   5.38 x 103cells per mL

Mean cell density of control at 72 hours                    :   1.10 x 106cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% 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) was3% 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 or test cultures at 1.0 mg/L. Some misshapen cells were observed to be present in the 3.2 and 10 mg/L test cultures whilst much cell debris was observed to be present in the 32 and 100 mg/L test cultures.

 

Water Quality Criteria

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

 

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.8 at 0 hours to pH 8.5 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, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were very pale green dispersions, the 32 mg/L test cultures were extremely pale green cultures whilst the 100 mg/L test cultures were clear colorless solutions.

Cell Densities and pH Values in the Definitive Test

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

7.8

5.73E+03

2.62E+04

1.98E+05

1.24E+06

8.5

 

R2

5.10E+03

2.45E+04

1.65E+05

9.61E+05

 

R3

6.16E+03

2.40E+04

1.74E+05

1.01E+06

 

R4

4.80E+03

2.71E+04

2.00E+05

1.24E+06

 

R5

5.20E+03

2.46E+04

1.93E+05

1.23E+06

 

R6

5.28E+03

2.50E+04

1.71E+05

9.39E+05

 

Mean

5.38E+03

2.52E+04

1.83E+05

1.10E+06

1.0

R1

7.8

4.35E+03

2.28E+04

1.76E+05

1.05E+06

8.4

 

R2

4.06E+03

2.50E+04

1.71E+05

1.06E+06

 

R3

4.42E+03

2.60E+04

1.61E+05

1.05E+06

 

Mean

4.28E+03

2.46E+04

1.69E+05

1.06E+06

3.2

R1

7.7

5.78E+03

2.56E+04

1.36E+05

7.24E+05

8.3

 

R2

6.08E+03

2.55E+04

1.51E+05

9.03E+05

 

R3

5.68E+03

2.07E+04

1.10E+05

6.82E+05

 

Mean

5.85E+03

2.39E+04

1.32E+05

7.70E+05

10

R1

7.7

5.20E+03

1.12E+04

4.19E+04

1.82E+05

7.9

 

R2

6.27E+03

9.65E+03

2.73E+04

1.01E+05

 

R3

6.05E+03

9.49E+03

2.13E+04

6.84E+04

 

Mean

5.84E+03

1.01E+04

3.02E+04

1.17E+05

32

R1

7.6

6.10E+03

6.82E+03

7.06E+03

1.03E+04

7.9

 

R2

5.26E+03

6.59E+03

5.41E+03

1.01E+04

 

R3

6.14E+03

7.15E+03

6.96E+03

9.01E+03

 

Mean

5.83E+03

6.85E+03

6.47E+03

9.78E+03

100

R1

7.6

6.43E+03

6.56E+03

6.86E+03

6.77E+03

7.8

 

R2

5.34E+03

5.84E+03

5.86E+03

5.76E+03

 

R3

5.90E+03

6.27E+03

5.70E+03

4.54E+03

 

Mean

5.89E+03

6.22E+03

6.14E+03

5.69E+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

.

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 based on the geometric mean measured test concentrations:

Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate 9.5 7.4 - 12 2.5 8.1
Yield 3.5 2.9 - 4.2 0.76 2.5
Executive summary:

Introduction

A study was perford to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. 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 subcapitatawas exposed to an aqueous of the test item at concentrations of 1.0, 3.2, 10, 32 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

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 87% to 95% of nominal. A slight decline in measured test concentration was observed at 72 hours in the range of 67% to 84% 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 ofPseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations: 

Response Variable

EC50(mg/L)

95% Confidence Limits (mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Lowest Observed Effect Concentration (LOEC) (mg/L)

Growth Rate

9.5

7.4

-

12

2.5

8.1

Yield

3.5

2.9

-

4.2

0.76

2.5

Description of key information

For aquatic algae, the 72-h ErC50 and NOEC values are 9.5 mg/L and 2.5 mg/L, respectively, as determined from a study according to OECD TG 201 and in compliance with GLP criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.5 mg/L
EC10 or NOEC for freshwater algae:
2.5 mg/L

Additional information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, freshwater alga (P. subcapitata, 3 replicates per treatment group) at a nominal starting cell concentration of ca. 5E+05 cells/mL were exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions. Nominal test concentrations were verified by a validated analytical method. The measured concentrations at the start of the test were between 87 and 95% of nominal but declined to 76 to 89% of initial concentrations at the end of the test. Effect values were therefore expressed as geomean measured concentrations (0.76, 2.5, 8, 27 and 90 mg/L for nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, respectively). Cell densities were determined using a particle counter at 0, 24, 48 and 72 hours after exposure and growth rate and biomass calculated. In addition, at 72 hours the shape and size of the algal cells was inspected microscopically and any abnormalities recorded. All OECD validity criteria were met.

Microscopic investigation revealed no abnormalities in the control or test cultures at 1.0 mg/L. Some misshapen cells were observed to be present in the 3.2 and 10 mg/L test cultures whilst much cell debris was observed to be present in the 32 and 100 mg/L test cultures. Based on cell counts inhibition of growth rates of 1, 7, 73, 88 and 98% were observed at the geomean test concentrations of 0.76, 2.5, 8, 27 and 90 mg/L, respectively. Based on these findings, the 72-h ErC50 and ErC10 values were determined at 9.5 and 2.9 mg/L, respectively. The 72 -h NOErC was determined to be 2.5 mg/L.