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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July 2016 to 12 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
CAS RN: 68412-26-0
Purity: This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB).
Description: Yellow powder
Analytical monitoring:
yes
Details on sampling:
Samples from the control and the 100 mg/L loading rate WAF test groups were taken from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis,, in duplicate. All samples were stored frozen prior to analysis. Two additional samples of the control and 100 mg/L loading rate WAF prepared at 0 hours were incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Preparation of Calibration Standards
The test item (nominal 20 mg) was dissolved in acetonitrile (200 mL) to prepare a stock solution with a nominal concentration of 100 mg/L. This stock solution was further diluted with acetonitrile to obtain a nominal 1.0 mg/L calibration standard. A duplicate calibration standard was similarly prepared at 1.0 mg/L. These duplicate calibration standards were used to determine the recovery and test sample concentrations.

Preparation of Test Samples
The test samples were thawed in a water bath. An aliquot (0.5 mL) of formic acid was added to the test sample samples. The test item was extracted from the test samples using a solid phase extraction cartridge (strata C8, 500 mg/ 3 mL). The cartridge was pre-conditioned with 10 mL of acetonitrile and 10 mL of water. The samples were drawn through the cartridge under reduced pressure. Samples containing algal cells were filtered through a plug of glass wool situated at the top of the cartridge. Subsequently, the cartridge was eluted with 10 mL of acetontrile into a 10 mL volumetric and made up to the mark with acetonitrile.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

A nominal amount of test item (250 mg) was added to the surface of 2.5 liters of culture medium to give the 100 mg/L loading rate. 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 mixture allowed to stand for 1 hour. Visual observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). 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. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no undissolved test item remained in the water column.

An aliquot (2 liters) of the WAF was inoculated with algal suspension (26.3 mL) to give the required test concentration of 100 mg/L loading rate WAF.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 ºC.

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 10^3 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 ºC until the algal cell density was approximately 10^4 – 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1 ºC
pH:
The pH value of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 8.0 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 specified in the Test Guidelines.
Nominal and measured concentrations:
Nominal: 100 mg/L
Measured: Less than the limit of quantification (0.0067 mg/L)
Details on test conditions:
As in the range-finding test, six 250 mL glass conical flasks each containing 100 mL of test preparation were used for the control and 100 mg/L loading rate WAF treatment groups.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.80 x 10^5 cells per mL. Inoculation of 2 liters of test medium with 26.3 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 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:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Details on results:
Analysis of the test solutions at 0, 24, 48 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed, determined to be 0.0067 mg/L were obtained. This does not infer that no test item was in solution, only that any present was at a concentration of less than the LOQ.

Given that the toxicity in this study 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 72-hour EL50 values, for growth rate, yield and biomass of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.
Results with reference substance (positive control):
Exposure conditions and data evaluation for the positive control, potassium dichromate 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): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 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.

Inhibition of Growth Rate, Yield and Biomass 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

0.066

-

5.90E+05

-

R2

0.067

-

6.22E+05

-

R3

0.068

-

6.60E+05

-

R4

0.066

-

5.77E+05

-

R5

0.066

-

5.73E+05

-

R6

0.067

-

6.14E+05

-

Mean

0.067

-

6.06E+05

-

SD

0.001

-

3.29E+04

-

100 mg/ L

R1

0.068

[1]

6.60E+05

-

R2

0.066

1

5.94E+05

-

R3

0.066

1

5.77E+05

-

R4

0.068

[1]

6.54E+05

-

R5

0.066

1

5.79E+05

-

R6

0.068

[1]

6.43E+05

-

Mean

0.067

0

6.18E+05

[2]

SD

0.001

 -

3.87E+04

 -

 

 

Nominal Loading Rate
(mg/L)

Biomass Integral (cells/mL)

0 – 72 h

% Inhibition

Control

R1

1.01E+07

-

R2

1.08E+07

-

R3

1.13E+07

-

R4

9.99E+06

-

R5

9.66E+06

-

R6

1.06E+07

-

Mean

1.04E+07

-

SD

5.84E+05

-

100 mg/ L

R1

1.17E+07

[12]

R2

1.01E+07

3

R3

1.02E+07

2

R4

1.09E+07

[5]

R5

9.78E+06

6

R6

1.10E+07

[6]

Mean

1.06E+07

[2]

SD

7.01E+05

 

* In accordance with the OECD test guideline 201, only the mean value for yield is calculated

R1 - R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth compared to control]

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata was investigated and the 72-hour EL50 values based on growth rate, yield and biomass were determined to be greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was 100 mg/L loading rate WAF.
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 was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) 761/2009.

Due to the low aqueous solubility and complex nature of the test item, the test solutions were prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 ºC.

Samples of the test solutions were removed daily and cell concentrations were determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the test solutions at 0, 24, 48 and 72 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed, determined to be 0.0067 mg/L were obtained. This does not infer that no test item was in solution, only that any present was at a concentration of less than the LOQ.

Given that the toxicity in this study 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 resulted in 72-hour EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata was investigated and the 72-hour EL50 values based on growth rate, yield and biomass were determined to be greater than 100 mg/L loading rate WAF.  The No Observed Effect Loading rate (NOEL) was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information