Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

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:
01 November 2018 - 24 June 2019
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
Specific details on test material used for the study:
Purity: 100% (UVCB)
Appearance/Physical state: Solid, white pastille
Storage: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each test group at 0 hours and at 72 hours for immediate quantitative analysis. A set of duplicate samples were taken at each occasion and stored frozen for further analysis if necessary. Samples of 5 mL of each test preparation were incubated alongside the test vessels to provide 24 and 48 hour samples. These samples were frozen before analysis.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a WAF of the test item based on methods established during preliminary media preparation trials.

Nominal amounts of test item (2.5, 5, 10, 20 and 40 mg) were each separately added to the surface of 2 liters of culture medium to achieve the 1.25, 2.5, 5, 10 and 20 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a slow stirring rate such that no vortex was formed at the water surface. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1 hour. A significant amount of dispersed test item was observed in the water column, so the WAFs were filtered through a glass wool plug (2 to 4 cm). 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 to 100 mL filtered was discarded) to give the 1.25, 2.5, 5, 10 and 20 mg/L loading rate WAFs.

Microscopic observations of the WAFs were performed, after filtering through the glass wool plug and one sheet of filter paper, which showed micro-dispersions of test item to be present in the 5, 10 and 20 mg/L loading rate WAFs. No micro-dispersions of test item were present in the 1.25 and 2.5 mg/L loading rate WAFs. Following further filtration through a second sheet of filter paper, micro-dispersions of test item were observed in the 10 and 20 mg/L loading rate WAFs but no micro-dispersions of test item were observed in the 1.25, 2.5 and 5.0 mg/L loading rate WAFs. All WAF preparations were treated with the same filtration regime in order to ensure consistency in the preparation method even if no micro-dispersions were observed.

An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.05 mL) to achieve the nominal loading rates.

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, 1.25, 2.5, 5 and 10 mg/L loading rate WAF test cultures were observed to be green dispersions, and the 20 mg/L loading rate WAF test cultures were observed to be light green dispersions.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: 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 maintained in the laboratory with periodic replenishment of the culture medium.
- Culturing media and conditions (same as test or not): Culture medium was the same as used in the rangefinding and definitive tests. Cultures were maintained under constant agitation by orbital shaker (150 rpm) and constant illumination at 24 +/- 1 deg C.
- Any deformed or abnormal cells observed: none reported
- Pre-culture cell density: 8.20 x 105 cells per mL; in log phase growth
Test type:
static
Water media type:
other: algal medium
Remarks:
; prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1with 0.1N NaOH or HCl.
Total exposure duration:
72 h
Test temperature:
24 +/- 1 deg C
pH:
7.5 - 7.9
Nominal and measured concentrations:
Nominal WAF loading rates: 0 (negative control), 1.25, 2.5, 5, 10 and 20 mg/L
Airthmetic mean measured concentrations:
The definitive test for this study was conducted with a loading rate in excess of the limit of solubility of the test item to ensure dose levels up to the functional solubility under the conditions of the test. Given that the toxicity cannot be attributed to a single component or a specific mixture of components that may have been present in the WAF solutions, the results were based on nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks, containing 100 mL of test medium, plugged with polyurethane foam bungs
- Aeration: constantly shaken at approximately 150 rpm
- Initial cells density: 5.00 x 10^3 cells per mL (nominal)
- Control end cells density: 8.49 x 10^5 cells per mL
- No. of vessels per concentration (replicates): three *
- No. of vessels per control (replicates): six
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: The pH of the control and each test concentration was determined at 0 and 72 hours. The temperature within the incubator and appearance of the test media were recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: continuous
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples taken at 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter. Three determinations were made for each sample.
- Other: The shape and size of the algal cells was inspected microscopically at test termination (72 hours).

[* During the incubation period, Replicate 2 of the 20 mg/L loading rate WAF test group detached from the shaker platform and broke, resulting in only two replicates for this group.]
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 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.:
10 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.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
7.7 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
20 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
other: yield
Details on results:
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 for 1.25, 2.5 and 5 mg/L loading rate WAFs; however cell debris and some healthy cells were observed to be present in the test cultures at 10 and 20 mg/L loading rate WAFs.
Results with reference substance (positive control):
A positive control test (Envigo Study Number YJ31TQ; 12 November - 03 December 2018), using potassium dichromate as the reference item, was conducted 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 to 72 hour): 1.5 mg/L (95% C.I.: 1.3 to 1.7 mg/L)
EyC50 (0 to 72 hour): 0.76 mg/L (95% C.I.: 0.69 to 0.85 mg/L)
NOEC (growth rate and yield): 0.50 mg/L
LOEC (growth rate and yield): 1.0 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:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1: Cell Densities and Percentage Inhibition of Growth from the Range‑finding Test

Nominal Loading Rate

(mg/L)

Cell Densities* (cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

4.46E+03

1.19E+05

-

-

R2

3.84E+03

4.84E+05

Mean

4.15E+03

3.01E+05

2

R1

4.58E+03

8.88E+05

[25]

[211]

R2

3.81E+03

9.70E+05

Mean

4.19E+03

9.29E+05

20

R1

4.93E+03

7.87E+05

[17]

[157]

R2

4.93E+03

7.50E+05

Mean

4.93E+03

7.69E+05

* Cell densities represent the mean number of cells per mL calculated from three cell counts for each of the replicate flasks.

R: Replicate

[ ]  Increase in growth compared to controls

-  Not applicable

Table 2: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.075

-

1.09E+06

-

R2

0.071

8.19E+05

R3

0.070

7.42E+05

R4

0.068

6.69E+05

R5

0.072

8.80E+05

R6

0.072

8.65E+05

Mean

0.071

8.44E+05

DS

0.002

1.44E+05

1.25

R1

0.075

[6]

1.12E+06

 

R2

0.073

[3]

9.33E+05

 

R3

0.071

0

8.48E+05

 

Mean

0.073

[3]

9.67E+05

[15]

SD

0.002

-

1.40E+05

 

2.5

R1

0.075

[6]

1.11E+06

 

R2

0.073

[3]

9.61E+05

 

R3

0.071

0

8.43E+05

 

Mean

0.073

[3]

9.72E+05

[15]

SD

0.002

-

1.34E+05

 

5

R1

0.074

[4]

1.01E+06

 

R2

0.070

1

7.62E+05

 

R3

0.069

3

7.16E+05

 

Mean

0.071

0

8.28E+05

2

SD

0.003

-

1.55E+05

 

10

R1

0.071

0

8.52E+05

 

R2

0.065

8

5.50E+05

 

R3

0.067

6

6.06E+05

 

Mean

0.068

5

6.69E+05

21

SD

0.003

-

1.61E+05

 

20

R1

0.064

10

4.92E+05

 

R2

-

-

-

 

R3

0.059

17

3.38E+05

 

Mean

0.062

14

4.15E+05

51

SD

0.004

-

1.09E+05

 

 * In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R: Replicate

SD: Standard deviation

[ ]  Increase in growth compared to controls

-  Not applicable

Validity criteria fulfilled:
yes
Remarks:
All validation criteria were met with the controls. Cell concentrations increased 170-fold after 72 hours; mean section-by-section coefficient of variation (CoV) for specific growth rate was 6%; CoV for average specific growth rate (0-72 h) was 3%.
Conclusions:
Following exposure of Pseudokirchneriella subcapitata to the test item for 72-hours, the EC50 values for growth rate and yield were determined to be greater than 20 and 19 mg/L nominal loading rate WAF, respectively. The No Observed Effect Loading Rate (NOEL) and Lowest Observed Effect Loading Rate (LOEL) were determined to be 10 mg/L and 20 mg/L, respectively.
Executive summary:

A study was conducted performed according to OECD Guidelines for Testing of Chemicals No. 201 and EC Method C.3 to assess the effects of the substance on the growth of the green alga, Pseudokirchneriella subcapitata, following a 72-hour exposure. 

Due to the low aqueous solubility and complex nature of the substance, the test medium was prepared as a Water Accommodated Fraction (WAF). In preliminary media preparation trials, visible undissolved substance was observed in loading rates in excess of 20 mg/L after filtering through glass wool and two sheets of filter paper. It was therefore considered unnecessary and unrealistic to test at loading rates in excess of 20 mg/L loading rate WAF.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.25, 2.5, 5, 10 and 20 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/-1 deg C. A control group was maintained concurrently under identical conditions, but was not exposed to the substance.

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

The substance concentration in the test samples collected at 0, 24, 48 and 72 hours was determined by high performance liquid chromatography with mass spectrometry (HPLC-MS) using an external standard. Quantitation was based on a single peak attributed to the major component of the test item. Analysis of the test preparations at 0 hours showed that measured test concentrations ranged from 0.0022 to 0.67 mg/L. At 72 hours, measured concentrations ranged from less than the Limit of Quantification (LOQ, 0.000516 mg/L) to 0.0095 mg/L. Given that the toxicity cannot be attributed to a single component or a specific mixture of components, the results were based on nominal loading rates WAF.

Following exposure of Pseudokirchneriella subcapitata to the test item for 72-hours, the EL50 values for growth rate and yield were determined to be greater than 20 and 19 mg/L nominal loading rate WAF, respectively. The No Observed Effect Loading Rate (NOEL) and Lowest Observed Effect Loading Rate (LOEL) were determined to be 10 mg/L and 20 mg/L, respectively.

Description of key information

A study was conducted according to OECD Guideline 201 and EC Method C.3, under GLP conditions, to assess the effects of the substance on the growth of the freshwater green alga, Pseudokirchneriella subcapitata. Exposure of Pseudokirchneriella subcapitata to the substance for 72 hours resulted in an ErL50 of greater than 20 mg/L, ErC10 of 17 mg/L, NOEC of 10 mg/L, and a LOEL of 20 mg/L loading rate WAF, based on growth rate and nominal loading rates WAF.

Key value for chemical safety assessment

Additional information