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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 December 2010 to 22 December 2010
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
April 1996, “Public Draft”
Qualifier:
according to guideline
Guideline:
other: JMAFF Test Guidelines (Algae growth inhibition)
Version / remarks:
2005
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled. All samples were deep-frozen immediately after sampling and stored at about -20 °C until analysis. In pre-experiments for investigation of the storage stability of the samples (nonGLP), the sample proved to be stable under these storage conditions. The concentrations of the test substance were determined in one test medium sample per test concentration from both sampling dates (0 and 96 hours). From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times. The algae were separated from the samples by filtration prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Reconstituted test water (OECD medium) prepared according to OECD test guideline 201 was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the test water.
Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 50.17 mg of the test item in 500 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 72 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. The stirring period of 72 hours was chosen according to the results of a pre-experiment (without GLP) which showed that the solution equilibrium was reached after this time. After the 72- hour stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.45 µm). The undiluted filtrate was used as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was serially diluted with test water. The test media were prepared just before the start of the test (= start of exposure). The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Method of cultivation: An inoculum culture was set up three days before the start of the exposure. The algae were kept in the exponential growth phase until inoculation of the test solutions.
- Culturing conditions same as test or not: Same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
24 mg/L as CaCO3
Test temperature:
22 °C
pH:
- Test start: 8.2
- Test end: 8.2 - 8.8
Nominal and measured concentrations:
- Nominal loading rate: 1:1600, 1:500, 1:160, 1:50, 1:16 and 1:5 of the undiluted filtrate with the loading rate of 100 mg/L
- Measured concentration: 0.052, 0.152, 0.527,1.56, 5.16 and 15.4 mg/L, respectively
- During the test period of 96 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 65 to 91% of the initially measured concentrations were found. See Table 1 in "Any other information on materials and methods incl.tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer with 15mL of test solution
- Type: closed (covered with a glass dish)
- Mixing condition: During the test, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes
- Composition of the medium: See Table 2 in "Any other information on materials and methods incl. tables"

TEST MEDIUM / WATER PARAMETERS
- pH: The pH was measured and recorded in each treatment at the start and end of the test.
- Temperature: The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
- Appearance: The appearance of the test media was also recorded daily. Additionally, the water temperature was continuously recorded with a data logger.

OTHER TEST CONDITIONS
- Light intensity and quality: Fluorescent tubes, approximately 5000 Lux (4520 - 5400 Lux). The light intensity over the incubation area (measured at nine places in the experimental area) was within a ±15%-deviation from the average light intensity.
- Light location: Above the test flasks

EFFECT PARAMETERS MEASURED:
- Determination of algal biomass: A small volume of the algal suspension was withdrawn from each test flask daily for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement ( Multi-Detection Microplate Reader, Model FLx800). The measurements were performed in duplicate. At the end of the test, a sample was taken from the control and from the test concentration of Dilution 1:160. The shape and size of the algal cells were examined microscopically in these samples. This test concentration was chosen, since at the three highest nominal concentrations of Dilution 1:5, 1:16 and 1:50 the algal cell density was too low for a reliable examination.

RANGE FINDING STUDY
- The selection of the test concentrations was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test substance in the test water (non-GLP).
- The enlarged spacing factor of 3.2 between the test concentrations was chosen based on the results of the range-finding test that documented a rather flat concentration-effect relationship, which is why a large concentration range had to be tested.
Reference substance (positive control):
yes
Remarks:
Not carry out with the main study (see "Results with reference substance (positive control)")
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.46 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% C. L.: 6.6 - 13 mg/ L
Details on results:
Overview of the results are provided in Table 3 - Table 6 at "Any other information on results incl. tables"
DENSITY
The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 5.0E+03 algal cells/mL, corresponding to 0.96E+03 relative fluorescence units.
Following 72-hours of exposure, cell biomass in the control averaged 9.02 x 104 in fluorescence units. The 72-hour averaged biomass in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels were 9.54, 8.23, 5.07, 3.73, 3.27 and 0.40E+04 in fluorescence units, respectively. Following 96-hours of exposure, cell biomass in the control averaged 2.548E+05 in fluorescence units. The 96-hour averaged biomass in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels were 2.868, 2.225, 1.416, 1.025, 0.711, 0.055E+05 in fluorescence units, respectively.

AUGC
Following 72-hours of exposure, AUGC in the control averaged 2.359E+05 x day in fluorescence units. Following 72-hours of exposure, cell biomass in the control averaged 6.44E+04 in fluorescence units. The 72-hour averaged biomass in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels were 6.82, 5.96, 3.97, 3.23, 2.81 and 0.62E+04 in fluorescence units, respectively. The 96-hour averaged biomass in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels were 2.584, 2.110, 1.349, 1.013, 0.795 and 0.100E+05 x day in fluorescence units, respectively.
Based on the results of Welch t - test (one-sided, α = 0.05), at 72 hours, a significant reduction in AUGC was detected in the 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 0.13 mg/L. The 72-hour EbC50 was determined to be 1.6 mg/L (with corresponding 95% confidence interval 1.1 – 2.3 mg/L). At 96 hours, a significant reduction AUGC was detected in the 0.13 mg/L and at all higher test concentrations, compared to the control data. Therefore, the 96-hour NOEbC for biomass was determined to be 0.042 mg/L. The 96-hour EbC50 was 1.0 mg/L (with corresponding 95% confidence interval 0.78 – 1.4 mg/L).

YIELD
The 72-hour yield in the control averaged 8.93E+04 cells/mL, The 72-hour averaged biomass in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels were 9.44, 8.14, 4.98, 3.63, 3.18 and 0.30E+04 cells/mL, respectively. The 96-hour yield in the control averaged 8.93E +04 cells/mL, The 96-hour averaged biomass in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels were 2.539,2.859, 2.215, 1.407, 1.016, 0.709, 0.045E+05 cells/mL, respectively.
Based on the results of Williams t-test (one-sided, α = 0.05), at 72 hours, a significant reduction in yield was detected in the 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEyC was determined to be 0.13 mg/L. The 72-hour EyC50 was determined to be 1.0 mg/L (with corresponding 95% confidence interval 0.72 – 1.5 mg/L). At 96 hours, a significant reduction in yield was detected in the 0.13 mg/L and at all higher test concentrations, compared to the control data. Therefore, the 96-hour NOEbC for biomass was determined to be 0.042 mg/L. The 96-hour EbC50 was 0.87 mg/L (with corresponding 95% confidence interval 0.67 – 1.1 mg/L).

GROWTH RATE
Following 72 hours of exposure, growth rate in the control averaged 1.51 days-1. The 72-hour growth rate in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7mg/L treatment levels averaged 1.53, 1.48, 1.32, 1.22, 1.18 and 0.47days-1, respectively. Following 96 hours of exposure, growth rate in the control averaged 1.40 days-1. The 96-hour growth rate in the 0.042, 0.13, 0.46, 1.37, 4.63 and 14.7mg/L treatment levels averaged 1.43, 1.36, 1.2, 1.17, 1.08 and 0.43 days-1, respectively.
Based on the results of Welch t-test (one-sided, α = 0.05), at 72 hours, a significant reduction in growth rate was detected in the 1.37, 4.63 and 14.7 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC for growth rate was determined to be 0.46 mg/L. Based on the findings, the 72-hour ErC50 was determined to be 9.0 mg/L (with corresponding 95% confidence interval 6.6 – 13 mg/L).
Based on the results of Williams t-test (one-sided, α = 0.05), at 96 hours, a significant reduction in growth rate was detected in the 0.46, 1.37, 4.63 and 14.7 mg/L treatment levels, compared to the control data. Therefore, the 96-hour NOErC was determined to be 0.13mg/L. Based on the findings, the 96-hour ErC50 was determined to be 8.9 mg/L (with corresponding 95% confidence interval 7.1 - 12 mg/L).

MICROSCOPIC EXAMINATION
The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of Dilution 1:160 and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to at least this concentration. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
Results with reference substance (positive control):
Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in October 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.99 mg/L, range of the 72- hour EC50 for the growth rate from 2000 to 2010: 0.71-1.7 mg/L).
Reported statistics and error estimates:
AUC, growth rate and yield were calculated for each test flask. The mean values for AUC, growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data. The 72-hour and 96-hour EC10, EC20 and EC50 values for the inhibition of AUC, average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis. The AUC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test or Welch t-test.

Table 3. Summary of results for the toxicity of the test substance to Pseudokirchneriella subcapitataafter 72 and 96 hours (based on mean measured concentrtaions of the test item)

 

after 72 h

after 96 h

Parameter

AUC

Growth rate

Yield

AUC

Growth rate

Yield

EC50(mg/L)

1.6

9.0

1.0

1.0

8.9

0.87

95% CI

1.1-2.3

6.6-13

0.72-1.5

0.78-1.4

7.1-12

0.67-1.1

EC20(mg/L)

0.24

2.1

0.19

0.19

2.6

0.17

95% CI

0.12-0.39

1.1-3.0

0.09-0.30

0.11-0.28

1.7-3.5

0.11-0.25

EC10(mg/L)

0.090

0.96

0.076

0.077

1.4

0.074

95% CI

0.034-0.17

0.38-1.6

0.028-0.14

0.035-0.13

0.70-2.1

0.038-0.12

NOEC (mg/L)

0.13

0.46

0.13

0.042

0.13

0.042

LOEC (mg/L)

0.46

1.37

0.46

0.13

0.46

0.13

95% CI: 95% confidence interval

Table 4. Yield

 

Treatment / dilution

Mean measured concentration

Yield Y (x 103) and inhibition of Y (Iy)

0-24 h

0-48 h

0-72 h

0-96 h

(mg/L)

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Y

Iy(%)

Control

3.0

0.0

16.8

0.0

89.3

0.0

253.9

0.0

1:1600

0.042

3.1

-3.8

18.0

-7.0

94.4

-5.8

285.9

-12.6

1:500

0.13

2.9

2.8

16.0

4.7

81.4

8.8

221.5#

12.7

1:160

0.46

2.7

7.7

12.1*

28.1

49.8#

44.2

140.7#

44.6

1:50

1.37

2.8

5.0

11.3*

32.5

36.3#

59.3

101.6#

60.0

1:16

4.63

2.7

8.6

9.5*

43.1

31.8#

64.4

70.9#

72.1

1:5

14.7

2.1*

27.6

2.5*

84.9

3.0#

96.6

4.5#

98.2

*: mean value significantly lower than in the control (according to Welch t-test, one-sided, α = 0.05)

#: mean value significantly lower than in the control (according to Williams t-test, one-sided, α = 0.05)

Table 5. Areas Under the growth Curve (AUC).

 

 

Treatment / dilution

Mean measured concentration

Areas under the growth curves AUC (103*day) and inhibition of AUC (IAUC)

0-24 h

0-48 h

0-72 h

0-96 h

(mg/L)

AUC

IAUC (%)

AUC

IAUC (%)

AUC

IAUC (%)

AUC

IAUC(%)

Control

1.5

0.0

11.4

0.0

64.4

0.0

235.9

0.0

1:1600

0.042

1.5

-3.8

12.1

-6.2

68.2

-6.0

258.4

-9.5

1:500

0.13

1.4

2.8

10.9

4.2

59.6

7.5

211.0*

10.6

1:160

0.46

1.4

7.7

8.8*

22.8

39.7*

38.3

134.9*

42.8

1:50

1.37

1.4

5.0

8.5*

25.3

32.3*

49.8

101.3*

57.1

1:16

4.63

1.4

8.6

7.5*

34.1

28.1*

56.3

79.5*

66.3

1:5

14.7

1.1*

27.6

3.4*

69.9

6.2*

90.4

10.0*

95.8

*: mean value significantly lower than in the control (according to Welch t-test, one-sided,α= 0.05)

Table 6. Average growth rate (µ)

 

Treatment / dilution

Mean measured concentration

Average growth rate µ (1/day) and inhibition of µ (Ir)

0-24 h

0-48 h

0-72 h

0-96 h

(mg/L)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

µ

Ir(%)

Control

---

1.41

0.0

1.46

0.0

1.51

0.0

1.40

0.0

1:1600

0.042

1.44

-2.2

1.49

-2.5

1.53

-1.4

1.43

-2.2

1:500

0.13

1.39

1.3

1.44

1.3

1.48

1.9

1.36

2.3

1:160

0.46

1.35

4.1

1.30#

10.5

1.32

12.8

1.2#

10.7

1:50

1.37

1.37

2.6

1.28#

12.3

1.22*

19.3

1.17#

16.2

1:16

4.63

1.34

4.6

1.20#

17.8

1.18*

22.2

1.08#

22.6

1:5

14.7

1.18*

16.4

0.65#

55.6

0.47*

68.7

0.43#

68.9

*: mean value significantly lower than in the control (according to Welch t-test, one-sided, α = 0.05)

#: mean value significantly lower than in the control (according to Williams t-test, one-sided, α = 0.05)

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. tables'
Conclusions:
Based on the findings, the 72-hour ECr50 value was determined to be 9.0 mg/L (with corresponding 95% confidence interval 6.6 - 13 mg/L), and the 72-hour NOErC value was determined to be 0.46 mg/L.
Executive summary:

The influence of the test substance on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96-hour static test in accordance with OECD TG 201 and in compliance with GLP. Due to the low solubility of the test item in test water, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 72 hours. Subsequently, the dispersion was filtered. The dilutions 1:5, 1:16, 1:50, 1:160, 1:500 and 1:1600 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. Additionally, a control was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. At the start of the test, the analytically determined concentrations of the test substance in the test media (dilutions 1:1600, 1:500, 1:160, 1:50, 1:16 and 1:5) were 0.052, 0.152, 0.527, 1.56, 5.16 and 15.4 mg/L, respectively. During the test period of 96 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 65 to 91% of the initially measured concentrations were found. Every 24 hours, the biomass was determined by fluorescence measuring, on which the yield, AUC and growth rates were calculated. At the end of the study, algal cells were examined on appearance.


Based on the findings, the 72-hour ErC50 was 9.0 mg/L (with corresponding 95% confidence interval 6.6 - 13 mg/L), the EyC50 was 1.0 mg/L (with corresponding 95% confidence interval 0.72 – 1.5 mg/L) and the EbC50 was 1.6 mg/L (with corresponding 95% confidence interval 1.1 – 2.3 mg/L). The 96-hour ErC50 was 8.9 mg/L (with corresponding 95% confidence interval 7.1 - 12 mg/L), the EyC50 was 0.87 mg/L (with corresponding 95% confidence interval 0.67 – 1.1 mg/L) and the EbC50 was 1.0 mg/L (with corresponding 95% confidence interval 0.78 – 1.4 mg/L).


The LOEC at 72 hours, based on growth rate, was 1.37 mg/L, and based on yield and biomass integral was 0.46 mg/L. The LOEC at 96 hours, based on growth rate, was 0.46 mg/L, and based on yield and biomass integral was 0.13 mg/L. The NOEC at 72 hours, based on growth rate, was 0.46 mg/L, and based on yield and biomass integral was 0.13 mg/L. The NOEC at 96 hours, based on growth rate, was 0.13 mg/L, and based on yield and biomass integral was 0.042 mg/L.

Description of key information

All available data was assessed. The study following standard guidelines and with effect values based on growth rate was included here as the key study. The effect values are selected for the CSA. Other studies are included as supporting information. 


72-h NOErC = 0.46 mg/L and 72-h ErC 50 = 9.0 mg/L (–mean measured concentrations); Pseudokirchneriella subcapitata; freshwater species; OECD TG 201; Höger, 2011 


 

Key value for chemical safety assessment

EC50 for freshwater algae:
9 mg/L
EC10 or NOEC for freshwater algae:
0.46 mg/L

Additional information

There are four studies on freshwater algae and one study on marine water algae available for this endpoint. One of the studies followed standard guideline (OECD TG 201), was compliant with GLP criteria, and the effect values based on the algae growth rate. This study was selected as the key study. The other studies are included as supporting information.


In the key study, freshwater green algal Pseudokirchneriella subcapitata was investigated in a 96-hour static test. At the start of the test, the analytically determined concentrations of the test substance were 0.052, 0.152, 0.527, 1.56, 5.16 and 15.4 mg/L (dilutions 1:1600, 1:500, 1:160, 1:50, 1:16 and 1:5 of the undiluted filtrate with the loading rate of 100 mg/L, respectively). The test conditions were as follows: temperature 22 ˚C and pH 8.2 – 8.8. The 72-h ECr50 was determined to be 9.0 mg/L and NOErC was determined to be 0.46 mg/L, based on mean measured concentrations (Höeger 2011, GLP, Reliability 1). The other studies on freshwater algae showed that the EbC10 range from 0.016 to 6.78 mg/L and the EbC50 ranged from 0.093 to 13.58 mg/L (Hollister 1981 b, c, and d, no standard guidelines followed, GLP, Reliability 2). The effects on growth rate were not determined in these studies. In the study on marine diatom Skeletonema costatum, the 11d-EbC10 and EbC50 values were calculated to be < 18 µg/L and 21 µg/L, respectively, based on initial measured concentrations (Hollister 1981a, no standard guidelines followed, GLP, Reliability 2).


In addition, one study on green algae Dunaliella tertiolecta following ASTM is found in the RAC opinion of the test substance (December 2016). No detailed study record is available but the results are briefly summarized here. The 96 h ErC50 and NOErC were determined to be 2.33 and 0.375 mg/L, respectively (Baird and De Lorenzo 2010).