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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:
The study was conducted between 23 May 2016 and 30 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch number : 5399661C10
- Expiration date of the lot/batch: 12 January 2020
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:
Analytical monitoring:
yes
Remarks:
WAF determined
Details on sampling:
Range-finding test

A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis.

Definitive test

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
Vehicle:
no
Details on test solutions:
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.

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 nominal loading rates of 1.0, 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.

Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 liters of culture medium to give the 1.0, 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 1.0, 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 (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.1 mL) to give the required test concentrations of 1.0, 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 ± 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.

A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis.

Definitive Test

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
Nominal amounts of test item (10, 32, 20, 64 and 200 mg) were each separately added to the surface of 10, 10, 2, 2 and 2 liters of culture medium to give the 1.0, 3.2, 10, 32 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 1.0, 3.2, 10, 32 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 (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (9.4 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
:
- Method of cultivation: 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 illumination at 21 ± 2 °C.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 2 ”C
pH:
7.9 - 8.6
Nominal and measured concentrations:

Range-finding test

Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.023, 0.035 and 0.039 mg/L were obtained for the 1.0, 10 and 100 mg/L loading rate WAFs respectively. A decline in measured test concentrations was observed in all loading rate WAF test preparations at 72 hours to less than the limit of quantification (LOQ) which was determined to be 0.0019 mg/L suggesting that the test item was unstable over the test duration.

Definitive Test

Chemical Analysis of Test Loading Rates

Initial analysis of the 0-Hour test preparations showed measured test concentrations to range from 0.016 to 0.094 mg/L. A decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0019 mg/L, to 0.0022 mg/L. Recovery analyses run alongside the test sample analyses failed to meet the required validation criteria of a mean recovery value of greater than 80% at a nominal concentration of 0.020 mg/L. Given this, the duplicate 0-Hour test samples were analysed following the successful analysis of a further 5 recovery samples where an acceptable mean recovery rate of 89% was attained. Measured concentrations in the range of 0.024 to 0.094 mg/L were obtained from these duplicate samples. Due to the volume of test solution required for analysis, it was not possible to provide duplicate samples at 72 hours. As such, the reported values are based on analyses run alongside a mean recovery rate of 70%. This was considered to have had no adverse effect on the outcome of the test as, 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.
Details on test conditions:
Range-Finding Test

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 nominal loading rates of 1.0, 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.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.1 mL) to give the required test concentrations of 1.0, 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 ± 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.

A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. All samples were stored frozen prior to analysis.

Definitive Test

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
An aliquot (1 liter) of each of the loading rate WAFs was separately inoculated with algal suspension (9.4 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
ca. 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
ca. 6.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
>= 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Details on results:
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.

Observations on Test Item Solubility
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 colorless media columns with particles of test item floating at the media surface. Microscopic examination of the WAFs showed there to be no micro-dispersions of test item present.

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 loading rate WAF test cultures were observed to be green dispersions. The 10 mg/L loading rate WAF test cultures were pale green dispersions, the 32 mg/L loading rate WAF test cultures were very pale green dispersions whilst the 100 mg/L test cultures were clear colorless solutions.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 153 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 103 cells per mL
Mean cell density of control at 72 hours : 7.64 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 19% 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%.

Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Analytical analysis result of the test item concentrations

Results for Range-Finding Samples

 Time point (h)

 Nominal Loading Rate of

Test Item in Range-Finding Sample

Cnom (mg/mL)

 Sample Preparation Factor

F

 Determined Concentration of

Test Item in Range-Finding Sample

c (mg/mL)

 0

 1.0

10

100

0.10

0.10

0.10 

0.0230

0.0351

0.0387 

 72

1.0

10

100

0.10

0.10

0.10 

<LOQ

<LOQ

<LOQ

Results for Test Samples

 Time point (h)

 Nominal Loading Rate of

Test Item in Range-Finding Sample

Cnom(mg/mL)

 Sample Preparation Factor

F

 Determined Concentration of

Test Item in Range-Finding Sample

c (mg/mL)

 0

Control

1.0

3.2

10

32

100 

0.10

0.10

0.10

0.10

0.10

0.10 

<LOQ / 0.00296

0.0164 / 0.0240

0.0264 / 0.0291 

0.0531 / 0.0921

0.0626 / 0.0939

0.0939 / 0.0907

 72

Control

1.0

3.2

10

32

100

0.10

0.10

0.10

0.10

0.10

0.10 

<LOQ

<LOQ

<LOQ

<LOQ

0.00216

0.00221

Cell densities and percentage inhibition of growth

Range-Finding Test

 Nominal Loading Rate (mg/mL)     Cell Densities (cells/mL)     Inhibition Values (%)   
     0 Hours  72 Hours  Growth Rate  Yield
 Control  R1  7.01E+03  9.97E+05  -  -
   R2  7.15E+03  1.17E+06  -  -
   Mean  7.08E+03  1.09E+06  -  -
 1.0  R1  5.57E+03  1.22E+06  [4]  [2]
   R2  6.43E+03  9.98E+05    
   Mean  6.00E+03  1.11E+06  [4]  [2]
 10  R1  6.53E+03  6.82E+05    
   R2  7.39E+03  5.68E+05    
   Mean  6.96E+03  6.25E+03  11  43
 100  R1  5.92E+03  2.83E+05    
   R2  6.53E+03  2.79E+05    
   Mean  6.22E+03  2.81E+05  21  75

Definitive Test

 Nominal Loading Rate (mg/L)     pH  Cell densities (cells/mL)        pH
        0h  24h  48h  72h  72h
   R1    1.95E+04  1.44E+05  7.84E+05  
   R2    2.36E+04  1.70E+05  8.62E+05  
   R3    2.77E+04  1.91E+05  7.74E+05  
 Control  R4  8.1  2.02E+04  1.60E+05  7.14E+05  8.6
   R5    1.66E+04  1.17E+05  6.29E+05  
   R6    2.28E+04  1.90E+05  8.19E+05  
   Mean    2.17E+05  1.62E+05  7.64E+05  
   R1    2.59E+04  1.58E+05  7.65E+05  
   R2    2.24E+04  1.63E+05  9.62E+05  
 1.0  R3  8.1  2.59E+04  1.85E+05  9.73E+05  8.6
   Mean    2.47E+04  1.69E+05  9.00E+05  
   R1    2.25E+04  1.63E+05  8.23E+05  
   R2    1.21E+04  0.05E+05  5.54E+05  
 3.2  R3  7.9  1.05E+04  8.76E+04  5.96E+05  8.5
   Mean    1.50E+04  1.19E+05  6.58e+05  
   R1    8.74E+03  5.48E+04  2.75E+05  
   R2    5.50E+03

 3.97E+04

 2.21E+05  
 10  R3  8.0  5.30E+03  3.85E+04  2.54E+05  8.5
   Mean    6.51E+03  4.43E+04  2.50E+05  
   R1    3.76E+03  2.87E+04  1.92E+05  
   R2    8.64E+02  1.53e+04  1.29E+05  
 32  R3  8.0  5.28E+02  9.26e+03  8.43e+04  8.4
   Mean    1.72E+03  1.77E+04  1.35E+05  
   R1    2.86E+03  2.86e+04  1.82E+05  
   R2    9.92E+02  1.29E+04  8.64E+04  
 100  R3  7.9  1.41E+03  1.09E+04  1.10e+05  8.3
   Mean    1.75E+03  1.74E+04  1.26E+05  

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

0.083

0.071

 

R2

0.065

0.082

0.068

 

R3

0.071

0.080

0.058

 

R4

0.058

0.086

0.062

 

R5

0.050

0.081

0.070

 

R6

0.063

0.088

0.061

 

Mean

0.061

0.083

0.065

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

 0.070    7.82E+05

 

 

R2

0.072

 

8.57E+05

 

 

R3

0.070

 

7.69E+05

 

 

R4

0.069

-

7.09E+05

-

 

R5

0.067

 

6.24E+05

 

 

R6

0.071

 

8.14E+05

 

 

Mean

0.070

 

7.58E+05

 

 

SD

0.002

 

8.24E+04

 

1.0

R1

0.070

0

7.60E+05

 

 

R2

0.073

[4]

9.57E+05

 

 

R3

0.073

[4]

9.68E+05

 

 

Mean

0.072

[3]

8.95E+05

[18]

 

SD

0.002

1.17E+05

 

3.2

R1

0.071

[1]

8.18E+05

 

 

R2

0.065

7

5.49E+05

 

 

R3

0.066

6

5.91E+05

 

 

Mean

0.067

4

6.53E+05

14

 

SD

0.003

 

1.48E+05

 

10

R1

0.056

20

2.70E+05

 

 

R2

0.053

24

2.16E+05

 

 

R3

0.055

21

2.49E+05

 

 

Mean

0.055

22

2.45E+05

68

 

SD

0.002

2.72E+04

 

32

R1

0.051

27

1.77E+05

 

 

R2

0.045

36

1.24E+05

 

 

R3

0.039

44

7.93E+04

 

 

Mean

0.045

36

1.30E+05

83

 

SD

0.006

5.42E+04

 

100

R1

0.050

29

1.77E+05

 

 

R2

0.040

43

8.14E+04

 

 

R3

0.043

39

1.05E+05

 

 

Mean

0.044

37

1.21E+05

84

 

SD

0.005

4.98E+04

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 153 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 103 cells per mL

Mean cell density of control at 72 hours : 7.64 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 19% 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%.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:
Response Variable EL50 (mg/L Loading Rate WAF) No Observed Effect Loading Rate (NOEL) (mg/L) Lowest Observed Effect Loading Rate (LOEL) (mg/L)
Growth Rate >100 3.2 10
Yield 6.8 3.2 10
Executive summary:

Algal Growth Inhibiiton Test

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) 761/2009.

Methods… 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).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates 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

Initial analysis of the 0-Hour test preparations showed measured test concentrations to range from 0.016 to 0.094 mg/L. A decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0019 mg/L, to 0.0022 mg/L. Recovery analyses run alongside the test sample analyses failed to meet the required validation criteria of a mean recovery value of greater than 80% at a nominal concentration of 0.020 mg/L. Given this, the duplicate 0-Hour test samples were analysed following the successful analysis of a further 5 recovery samples where an acceptable mean recovery rate of 89% was attained. Measured concentrations in the range of 0.024 to 0.094 mg/L were obtained from these duplicate samples. Due to the volume of test solution required for analysis, it was not possible to provide duplicate samples at 72 hours. As such, the reported values are based on analyses run alongside a mean recovery rate of 70%. This was considered to have had no adverse effect on the outcome of the test as, 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.

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

Response Variable EL50 (mg/L Loading Rate WAF) No Observed Effect Loading Rate (NOEL) (mg/L) Lowest Observed Effect Loading Rate (LOEL) (mg/L) Growth Rate >100 3.2 10

Yield 6.8 3.2 10

Description of key information

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

Response Variable EL50 (mg/L Loading Rate WAF) No Observed Effect Loading Rate (NOEL) (mg/L) Lowest Observed Effect Loading Rate (LOEL) (mg/L) Growth Rate >100 3.2 10

Yield 6.8 3.2 10

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
3.2 mg/L

Additional information