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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:
04 August 2015 to 07 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None specified.
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter used to prepare the WSF was retained for analysis of the residue.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2.0 mL
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
The batch of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] tested was a white powder with a purity of 99.0 % and the substance was not soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test substance because, from an ecotoxicological point of view, the test substance was pure, i.e. the purity was ≥95%.
Preparation of test solutions started with a loading rate of 100 mg/L applying two days of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter (Whatman) where after the clear and colourless Water Soluble Fraction (WSF) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of E+04 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1e+4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period specified in the study report.
Hardness:
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 22 and 24°C.
Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
pH:
The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit).
Dissolved oxygen:
Not specified in the study report.
Salinity:
Not applicable - freshwater
Nominal and measured concentrations:
(1,2-Dioxoethylen)bis(iminoethylen) bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionat]: 1.0, 10 and 100% of the WSF prepared at a loading rate of 100 mg/L.
Details on test conditions:
Combined limit/range-finding test
Test concentrations: (1,2-Dioxoethylen)bis(iminoethylen) bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionat]: 1.0, 10 and 100% of the WSF prepared at a loading rate of 100 mg/L.
Control: Test medium without test substance or other additives
Replicates:
6 replicates each for the control and the highest test concentration;
3 replicates of each lower test concentrations;
1 extra replicate of the control and each test concentration for sampling purposes;
1 or 2 replicates of each test concentration without algae.

Test procedures and conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1e+4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 79 to 92 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the test microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.

Recording of cell densities
At the beginning of the test, cells were counted using a microscope and a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (path length =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
In the control and the highest test concentration cell densities were determined on 24 hour intervals. In the lower concentrations cell density was measured only at the end of the test.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 (Potassium dichromate)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate & yield
Remarks on result:
other: above the maximum solubility of the test substance in the test medium
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.5 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate & yield
Remarks on result:
other: above the maximum solubility of the test substance in the test medium.
Details on results:
Measured test substance concentrations
Samples taken from the highest test concentration were analysed and showed that the measured concentrations were below the lowest calibration standard of 0.2 μg/L. Analysis of the filter residue confirmed that the test substance was used for the preparation of the test solutions.

Inhibition of growth rate and inhibition of yield
No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Experimental conditions
The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 22 and 24°C.
Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).
Results with reference substance (positive control):
Pseudokirchneriella subcapitata, strain: NIVA CHL-1. Fresh water algal growth inhibition test with potassium dichromate (Project 509666).
Start: 29 June 2015
End: 02 July 2015
The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006; Annex 5 corrected 28 July 2011 and ISO Standard 8692, Second edition, 01 October 2004.
This reference test was carried out to check the sensitivity of the test system used by WIL Research Europe to Potassium dichromate (Merck, Art. 1.04864, Batch K44879664).
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0e+4 cells/ml.
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.5 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.46 mg/L with a 95% confidence interval ranging from 0.43 to 0.49 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested was within the low end of this range.
Reported statistics and error estimates:
None specified in the study report.

Percentage inhibition of growth rate (total test period)

Treatm.[% wsf 100 mg/l]

Mean

Std. Dev.

n

%inhibition

Control

1,00

10,00

100,00

1,442

1,458

1,439

1,505

0,0432

0,0154

0,0098

0,0377

6

3

3

6

 

-1,1

0,2

-4,4

 

Percentage inhibition of growth rate at different time intervals

Concentration, % WSF prep. at 100 mg/L

n

0 – 24h

24 – 48h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

100

6

6

1.32

1.51

0.0

-14.3

1.52

1.47

0.0

3.7

1.48

1.54

0.0

-3.9

 

Percentage inhibition of yield

Concentration, % WSF prep. at 10 mg/L

Mean

Std.Dev.

n

%Decrease

Control

1.0

10

100

75

78

74

91

9.96

3.63

2.21

9.99

6

3

3

6

 

-4.3

1.3

-21.1

 

pH levels

Concentration, % WSF prep. at 100 mg/L

pH

t=0h

t=72h

Control

100

8.0

8.0

8.0

7.9

 

Overview of % inhibition of growth rate in the reference test:

Nominal conc. K2Cr2O7(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

0.18

0.32

0.56

1.0

1.8

3.2

1.789

1.758

1.705

1.437

0.994

0.641

0.502

0.0217

0.0229

0.0414

0.0241

0.0430

0.0216

0.0333

3

3

3

3

3

3

3

0.0

1.7

4.7

19.7

44.5

64.2

71.9

 

Overview of % Inhibition of yield in the reference test:

Nominal conc. K2Cr2O7(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

0.18

0.32

0.56

1.0

1.8

3.2

213.756

194.553

166.401

73.570

18.822

5.850

3.530

13.7166

13.4084

20.9357

5.4959

2.6398

0.4397

0.4403

3

3

3

3

3

3

3

0.0

9.0

22.2

65.6

91.2

97.3

98.3

 

Validity criteria fulfilled:
yes
Conclusions:
Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] in water, concentration levels that might be toxic for algae could not be reached.
The measured concentration in the WSF prepared at a loading rate of 100 mg/L was below the lowest calibration standard of 0.2 μg/L.
The EC50 for inhibition of both growth rate (72h-ERC50) and yield (72h-EYC50) was above the maximum solubility of the test substance in the test medium.
The 72h-NOEC for inhibition of both growth rate and yield was at or above the maximum solubility of the test substance in the test medium.
Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat].

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the OECD series on testing and assessment number 23, 2000.

 

The batch of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] tested was a white powder with a purity of 99.0 % and the substance was not soluble in test medium at the loading rate initially prepared.

 

Preparation of test solutions started with a loading rate of 100 mg/L applying two days of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter where after the clear and colourless Water Soluble Fraction (WSF) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium.

 

Six replicates of exponentially growing algal cultures were exposed to a control and to the WSF prepared at a loading rate of 100 mg/L. In addition, three replicates per group were exposed to 1.0 and 10% of the WSF. The initial cell density was 104 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 of exposure and at the end of the test.

 

Samples taken from the highest test concentration were analysed and showed that the measured concentrations were below the lowest calibration standard of 0.2 μg/L. Analysis of the filter residue confirmed that the test substance was used for the preparation of the test solutions.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] in water, concentration levels that might be toxic for algae could not be reached.

 

The measured concentration in the WSF prepared at a loading rate of 100 mg/L was below the lowest calibration standard of 0.2 μg/L.

 

The EC50 for inhibition of both growth rate (72h-ERC50) and yield (72h-EYC50) was above the maximum solubility of the test substance in the test medium.

 

The 72h-NOEC for inhibition of both growth rate and yield was at or above the maximum solubility of the test substance in the test medium.

The substance is not considered to be hazardous to algae at the limit of solubility.

Description of key information

Study undertaken at a GLP accredited laboratory is accordance with OECD Guideline 201 and EU Method C.3.

Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] in water, concentration levels that might be toxic for algae could not be reached. The substance is not considered to be hazardous to algae at the limit of solubility.

Key value for chemical safety assessment

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

Additional information

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat].

The batch of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] tested was a white powder with a purity of 99.0 % and the substance was not soluble in test medium at the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/L applying two days of magnetic stirring to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter where after the clear and colourless Water Soluble Fraction (WSF) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium.

Six replicates of exponentially growing algal cultures were exposed to a control and to the WSF prepared at a loading rate of 100 mg/L. In addition, three replicates per group were exposed to 1.0 and 10% of the WSF. The initial cell density was 104 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 of exposure and at the end of the test.

Samples taken from the highest test concentration were analysed and showed that the measured concentrations were below the lowest calibration standard of 0.2 μg/L. Analysis of the filter residue confirmed that the test substance was used for the preparation of the test solutions.

 

Results

Due to the very low solubility of (1,2-Dioxoethylen)bis(iminoethylen)bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionat] in water, concentration levels that might be toxic for algae could not be reached.

The measured concentration in the WSF prepared at a loading rate of 100 mg/L was below the lowest calibration standard of 0.2 μg/L.

The EC50 for inhibition of both growth rate (72h-ERC50) and yield (72h-EYC50) was above the maximum solubility of the test substance in the test medium.

The 72h-NOEC for inhibition of both growth rate and yield was at or above the maximum solubility of the test substance in the test medium.

The substance is not considered to be hazardous to algae at the limit of solubility.