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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:
10 March 2017 - 25 April 2017
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)
Version / remarks:
23 March 2006; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
30 May 2008; Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23
Version / remarks:
14 December 2000
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Vapour pressure: 5.35 Pa at 24°C
- Solubility in water: 13.3 mg/L
Analytical monitoring:
yes
Details on sampling:
Single samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, 24, 48 and 72 h
Volume: 4.0 mL
Storage: Not applicable, samples were analysed on the day of sampling.

At the end of the exposure period, the replicates with algae were not pooled before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (10% saturated solution) but without algae. Samples for analysis were taken at the start of the test period and after 24, 48 and 72 hours of exposure.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of substance tested was a clear colourless liquid and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of 73 minutes (combined limit/range-finding test: 86 min.). Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 40 mL were added to each replicate vessel of the respective test concentration containing 0.80 mL of an algal suspension providing a cell density of 10^4 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Method of cultivation: 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 (60 to 120 µE/m2/s, in the range of 400-700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Pre-culture:
3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
Temperature was maintained between 22 and 23°C throughout the test.
pH:
t=0: 7.5
t=72 h: 7.4-7.9
Nominal and measured concentrations:
Combined limt/range-finding test: 1.0, 10 and 100% of a saturated solution prepared at 100 mg/L.

Final test:
Based on the results of the range-finding test the following concentrations were assigned to the final test: 1.0, 3.2, 10, 32 and 100% of a saturated solution prepared at 100 mg/L

Time-weighted average (TWA) test concentrations: 0.089, 0.22, 0.67, 1.1 (without algae), 2.6 and 8.9 mg/L. For more details see table 1 in field "Any other information on results'.

Since the concentrations did not remain stable over the entire exposure period and no concentration was determined for the sample taken after 24 hours of exposure, the effect concentrations are based on TWA concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL, air-tight closed vessels with minimal headspace to prevent loss of test substance due to volatilisation; fill volume: approx. 40 mL .
- The control group was maintained under identical conditions but not exposed to the test material.
- Initial cells density: 1 x 10^4 cells per mL
- Control end cells density: 16.53 x 10^4
- No. of vessels per concentration (replicates): 3 + 1 or 3 extra replicates of each test concentration for sampling purposes
- No. of vessels per control (replicates): 6
- Other: 2 extra replicates of each test concentration for sampling purposes after 24 and 48 hours of exposure.

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: adjusted M2 (prepared in accordance with OECD 201 using reverse osmosis purified deionised water (Milli-RO, Millipore)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 92 to 94 µE/m2/s.
- During the incubation, the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: 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 680 nm using a spectrophotometer with cuvettes (path length = 10 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the control and the 10% SS test group to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS IN RANGE FINDING STUDY
- Test concentrations: 1.0, 10 and 100% of a saturated solution prepared at 100 mg/L
- Results used to determine the conditions for the definitive study: yes, the results of the combined limit/range-finding test showed that expected EC50 for growth rate inhibition was between 10 and 100% of the staturated solution prepared at 100 mg/L.

TEST CONCENTRATIONS IN MAIN TEST
- Spacing factor for test concentrations: 3.2







Reference substance (positive control):
yes
Remarks:
Potassium dichromate (February 2017)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%-CI: 3.4-4.3 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.96 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95%-CI: 0.71-1.2 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.67 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities in algal cells: no

Results with reference substance (positive control):
- Results with reference substance valid? yes
- 72h-ErC50: 1.2 mg/L (95% confidence interval ranging from 1.1 to 1.2 mg/L)
- Other: 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.
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Williams Multiple Sequential t-test Procedure and Two-sample t-test Procedure, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined.
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Measured concentrations versus nominal concentrations:

At the start of the test, the actual test concentrations were 0.085, 0.28, 1.0, 3.5 and 12 mg/L in 1.0, 3.2, 10, 32 and 100% of the SS. These concentrations remained relatively stable during the first 48 hours of exposure, i.e. were at 79-135% of initial after 48 hours. At the end of the test, no test item concentration could be detected in the three lowest test concentrations, while 9.3-47% of the initial concentrations were measured in the two highest test concentrations.

During the combined limit/range-finding test and the final test, the concentrations measured in the samples taken from the solutions without algae were usually higher than the equivalent samples from solutions with algae. This indicates that sorption to the biomass may additionally explain the decline in measured concentrations aside from the volatile nature of the test item.

Due to technical problems with the gas chromatograph on the day of analysis, no concentrations were determined for the samples taken after 24 hours of exposure. The Time Weighted Average exposure concentrations were calculated based on the measurements on t=0, t=48 h and t=72 h (see table 1).

Table 1 Measured concentrations versus nominal concentrations

Substance concentration (mg/L)

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=48h

t=72 h

1.0

0.0853

no result

0.115

0.040b

0.089

3.2

0.279

no result

0.286

0.040b

0.22

10

1.03

no result

0.811

0.040b

0.67

10a

1.03

no result

1.17

0.799

1.1

32

3.53

no result

3.19

0.328

2.6

100

11.8

no result

8.33

5.60

8.9

awithout algae;

bDefined as (lowest calibration standard)/2.

Table 2 Growth rate and percentage inhibition for the total test period

TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.842

0.0223

6

0.089

1.829

0.0378

3

0.8

0.22

1.854

0.0244

3

-0.6

0.67

1.742

0.0429

3

5.4*#

2.6

1.176

0.2044

3

36.2*

8.9

0.392

0.0059

3

78.8*

* effect was statistically significant;

#effect was biologically not relevant (<10%)

Table 3 Growth rate and percentage inhibition at different time intervals

TWA conc. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.784

 

1.834

 

1.909

 

0.089

3

1.460

18.2

2.116

-15.4

1.909

0.0

0.22

3

1.706

4.4

1.894

-3.3

1.960

-2.7

0.67

3

1.636

8.3

1.748

4.7

1.843

3.5

2.6

3

1.398

21.7

0.584

68.2

1.545

19.1

8.9

3

1.053

41.0

-0.544

129.7

0.666

65.1

Table 4 Yield and percentage inhibition for the total test period

TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

250.9

16.53

6

0.089

241.2

27.83

3

3.9

0.22

259.5

19.39

3

-3.4

0.67

186.3

23.24

3

25.8*

2.6

37.6

23.76

3

85.0*

8.9

2.2

0.06

3

99.1*

* effect was statistically significant

Validity criteria fulfilled:
yes
Remarks:
In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%
Conclusions:
The ErC50, ErC10 and NOErC were 3.8, 0.96 and 0.67 mg/L, respectively.
Executive summary:

A study was performed to assess the effect of the substance on the growth of the green algae Pseudokirchneriella subcapitata. The test was performed according to OECD test guideline 201 and under GLP principles. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration and were 1.0, 3.2, 10 and 32% of the SS. The final test was performed using six exponentially growing algal cultures which were exposed to an untreated control and three replicates which were exposed to one of each test concentration. The exposure time was 72 hours.

Samples for analytical confirmation of actual exposure concentrations were taken at t=0, 24, 48 and 72 h. However, due to technical problems, no concentration was measured in the samples taken at t=24 h. This did not have a big implication on the quality and certainty of the study, because exposure concentrations remained stable in the first 48 hours (79 -135% of initial concentrations). Test item concentrations at the end of the test (t=72 h) could only be detected in the two highest concentrations and were 9.3 and 47% of initial. The lower concentrations were therefore determined to be equal to the lowest calibration standard divided by 2 (0.040 mg/L). The effect concentrations were based on the Time Weighted Average from the measured concentrations at t=0, 48 and 72 h.

The ErC50 was 3.8 mg/L with a 95% confidence interval of 3.4 -4.3 mg/L. The ErC10 was 0.96 mg/L with a 95% confidence interval of 0.71 -1.2 mg/L. The NOErC was 0.67 mg/L, at this concentration no biologically relevant inhibition of growth rate (< 10%) was seen. All acceptability criteria were met and the study was considered valid.

Description of key information

A study was performed to assess the effect of the substance on the growth of the green algaPseudokirchneriella subcapitata.The test was performed according to OECD test guideline 201 and under GLP principles. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration and were 1.0, 3.2, 10 and 32% of the SS. The final test was performed using six exponentially growing algal cultures which were exposed to an untreated control and three replicates which were exposed to one of each test concentration. The exposure time was 72 hours.

Samples for analytical confirmation of actual exposure concentrations were taken at t=0, 24, 48 and 72 h. However, due to technical problems, no concentration was measured in the samples taken at t=24 h. This did not have a big implication on the quality and certainty of the study, because exposure concentrations remained stable in the first 48 hours (79 -135% of initial concentrations). Test item concentrations at the end of the test (t=72 h) could only be detected in the two highest concentrations and were 9.3 and 47% of initial. The lower concentrations were therefore determined to be equal to the lowest calibration standard divided by 2 (0.040 mg/L). The effect concentrations were based on the Time Weighted Average from the measured concentrations at t=0, 48 and 72 h.

The ErC50 was 3.8 mg/L with a 95% confidence interval of 3.4 -4.3 mg/L. The ErC10 was 0.96 mg/L with a 95% confidence interval of 0.71 -1.2 mg/L. The NOErC was 0.67 mg/L, at this concentration no biologically relevant inhibition of growth rate (< 10%) was seen. All acceptability criteria were met and the study was considered valid.

Key value for chemical safety assessment

EC50 for freshwater algae:
3.8 mg/L
EC10 or NOEC for freshwater algae:
0.96 mg/L

Additional information