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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:
From April 6 - 22, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 4.6, 10, 22, 46 and 100 (% WSF of 100 mg/L loading rate)
- Sampling method: During the definitive test, singular samples for TOC-analysis were taken from all test groups (incubated without algae) according to the schedule presented below:
- Sampling frequency: 0, 24 and 72 h
- Sampling volume: 40 mL
- Sample storage: Samples were stored in a refrigerator until analyses

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with an aqueous mixture at a loading rate of 100 mg/L. This mixture was magnetically stirred for 48 h and then left to settle overnight. This resulted in a hazy dispersion with a floating layer and precipitate. Subsequently, the hazy water phase was collected by siphoning it through glass wool. This resulted in a slightly hazy solution that was used for testing. The lower test concentrations were prepared by subsequent dilution of the Water Soluble Fraction (WSF). The final test solutions were all clear and colourless except for the undiluted WSF that was slightly hazy. 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 10(4) cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture:
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm) in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): Yes (M2 media according to the OECD 201 was used)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21.8 and 23.9°C
pH:
7.4-8.1 (measured at the beginning and the end of the test)
Nominal and measured concentrations:
Measured concentration (Time weighted average): 0, 1, 1.7, 3.9 and 9.4 mg/L containing 0, 10, 22, 46 and 100% WSF (Water Soluble Fraction) , respectively. For 4.6% WSF, TWA concentration was not measured.
For details on measured concentrations versus nominal concentrations, please refer to 'table 1' in the 'Over all remarks' section
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: 100 mL glass vessel containing 50 mL of test solution
- Initial cells density: 10(4) cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration
- No. of vessels per control (replicates): 6 replicates of the control and 4 or 5 replicates of each test concentration without algae for sampling purposes (analytical monitoring).

GROWTH MEDIUM
- Standard medium used: Yes (M2)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water purified by reverse osmosis (Milli-RO)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination
- Light intensity and quality: Using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 63 to 73 E.m-2.s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.
- Other: At the end of the final test microscopic observations were performed on the test concentration closest to the EC50 to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Aprrox. 2
- Range finding study: Yes, six replicates of exponentially growing algae were exposed to a control and a WSF (Water Soluble Fraction) prepared at 100 mg/L.
- Test concentrations: Three replicates/concentration were exposed to dilutions containing 0.1, 1.0 and 10% of the WSF
- Others: Test procedure and conditions were similar to those applied in the final test with the following exceptions except: (1). pH was only measured in the control and the highest test concentration. (2). At the end of the test algae were not observed to verify a normal and healthy appearance (3) Samples for possible analysis were only taken from the control and the highest test concentration.
- Results used to determine the conditions for the definitive study: The final test was performed based on the results of the combined limit/range-finding test. For details on the results, please refer to 'table 1' and 'table 2' under 'Any other information on material and method incl. tables'.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 9.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: CI: 1.3-4.0 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
CI: 2.1-5.3 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- Growth rate: Growth rates were in the range of the controls at the TWA concentrations up to and including 1.0 mg/L during the 72 h test period, whereas the growth rate of algae exposed to 1.7 mg/L and higher were increasingly reduced with a maximum reduction of 40.6% at a TWA concentration of 9.4 mg/L. Statistically significant reduction of growth rate was found at TWA test concentrations of 1.7 mg/L and higher.

- Yield: Inhibition of yield increased with increasing concentration of test material from 1.7 mg/L upwards resulting in 87 % inhibition at the highest test concentration of 9.4 mg/L.. Statistically significant inhibition of yield was found at TWA test concentrations of 1.7 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- EC50:
1. The EC50 for growth rate reduction (ErC50: 0-72h) was 1.2 mg/L with a 95% confidence interval ranging from 0.77 to 2.0 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the ErC50 (0-72h) for the present batch corresponds with this range.
2. The EC50 for yield inhibition (EyC50: 0-72h) was 0.44 mg/L with a 95% confidence interval ranging from 0.37 to 0.54 mg/L. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the EYC50: 0-72h for the present batch corresponds with this range.
Reported statistics and error estimates:
Following statistical methods were employed:
- Chi-Square test for normality
- Levene's test for homogeneity of variance
- Bonferroni t-Test

Table1:  Mean cell densities (x104cells/mL) during the combined limit/range-finding test

Test group

 (% WSF of 100 mg/L loading rate)

Exposure time (h)

 

 

 

 

0

24

48

72

Control

1.0

5.5

27.1

161.5

0.10

1.0

5.3

24.2

147.4

1.0

1.0

5.0

21.7

148.9

10

1.0

5.1

22.7

145.4

100

1.0

3.0

9.9

32.0

Table 2: Percentage reduction of growth rate and inhibition of yield during the combined limit/range-finding test

Test group

 (% WSF of 100 mg/Lloading rate)

Mean growth rate

Yield (0-72 h)

 

 

 

 

µ (0-72 h)

Reduction (%)

x104cells/mL

Inhibition (%)

Control

0.07052

 

160.48

 

0.10

0.06931

1.7

146.35

8.8

1.0

0.06944

1.5

147.89

7.8

10

0.06914

2.0

144.44

10.0

100

0.04795

32.0

31.00

80.7

Table 3: Mean cell densities (x 104cells/mL) during the final test

Test group

(% WSF of

100 mg/L loading rate)

Test group

TWA conc.

(mg/l)

Exposure time (h)

 

 

 

 

0

24

48

72

Control

-

1.0

9.3

39.1

143.4

4.6

n.m.

1.0

9.4

42.0

147.4

10

1.0

1.0

8.6

41.1

139.9

22

1.7

1.0

6.0

28.7

104.5

46

3.9

1.0

5.5

11.9

62.6

100

9.4

1.0

3.8

5.3

19.5

Table 4: Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test

Test group

(% WSF of

100 mg/L loading rate)

Test group

TWA conc.

(mg/L)

Mean growth rate

Yield (0-72 h)

 

 

 

 

µ (0-72 h)

Reduction (%)

x104cells/mL

Inhibition (%)

Control

-

0.06896

 

142.40

 

4.6

n.m.

0.06932

-0.5

146.35

-2.8

10

1.0

0.06856

0.6

138.94

2.4

22

1.7

0.06450

6.5

103.55

27.3

46

3.9

0.05743

16.7

61.55

56.8

100

9.4

0.04098

40.6

18.46

87.0

Table 5: Percentage reduction of growth rate at different time intervals during the final test

Test group

(% WSF of

100 mg/L loading rate)

Test group

TWA conc.

(mg/L)

Mean growth rate

 

 

 

 

 

 

µ (0-24 h)

Reduction (%)

µ (24-48 h)

Reduction (%)

µ (48-72 h)

Reduction (%)

Control

-

0.09286

 

0.05993

 

0.05409

 

4.6

n.m.

0.09329

-0.5

0.06244

-4.2

0.05224

3.4

10

1.0

0.08934

3.8

0.06542

-9.2

0.05092

5.9

22

1.7

0.07428

20.0

0.06543

-9.2

0.05379

0.5

46

3.9

0.07084

23.7

0.03130

47.8

0.07015

-29.7

100

9.4

0.05387

42.0

0.01343

77.6

0.05563

-2.8

n.m. Not measured

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 hErC50 for growth rate reduction was > 9.4 mg/L, beyond the range tested and therefore above the maximum solubility of substance in test medium under the study conditions. The 72 h ErC10 was 2.3 mg/L (CI: 1.3 – 4.0 mg/L). The 72 h EbC50 was equivalent to 3.4 mg/L (CI: 2.1 - 5.3 mg/L) and the NOEC for both growth rate reduction and yield inhibition was 1.0 mg/L.
Executive summary:

A study was conducted to determine the toxicity of the test substance to freshwater green algae (Pseudokirchnerella subcapitata) according to OECD Guideline 201, in compliance with GLP. The substance was not completely soluble in test medium at the concentrations tested and hence the preparation included a 2 d magnetic stirring period followed by overnight settlement and subsequent collection of the Water Soluble Fraction (WSF). Exponentially growing algal cultures were exposed to 0, 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L. The total test period was 72 h and the initial algal cell density was 104 cells/mL. Samples for measurement of Total Organic Carbon (TOC) were taken at the start, then after 24 and 72 h of exposure. The time weighted average (TWA) exposure concentrations were calculated to correspond to 1.0, 1.7, 3.9 and 9.4 mg/L for the test groups containing 10, 22, 46 and 100% WSF, respectively. Growth rate and yield were significantly inhibited at a concentration of 1.7 mg/L (TWA) and higher. Under the study conditions, the 72 h ErC50 for growth rate was > 9.4 mg/L, beyond the range tested and therefore above the maximum solubility of substance in test medium under the study conditions. The 72 h ErC10 was 2.3 mg/L (CI: 1.3 – 4.0 mg/L). The 72 h EbC50 was equivalent to 3.4 mg/L (CI: 2.1 - 5.3 mg/L) and the NOEC for both growth rate and yield was 1.0 mg/L (Migchielsen, 2010).

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
9.4 mg/L
EC10 or NOEC for freshwater algae:
1 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to freshwater green algae (Pseudokirchnerella subcapitata) according to OECD Guideline 201, in compliance with GLP. The substance was not completely soluble in test medium at the concentrations tested and hence the preparation included a 2 d magnetic stirring period followed by overnight settlement and subsequent collection of the Water Soluble Fraction (WSF). Exponentially growing algal cultures were exposed to 0, 4.6, 10, 22, 46 and 100% of a WSF prepared at a loading rate of 100 mg/L. The total test period was 72 h and the initial algal cell density was 104 cells/mL. Samples for measurement of Total Organic Carbon (TOC) were taken at the start, then after 24 and 72 h of exposure. The time weighted average (TWA) exposure concentrations were calculated to correspond to 1.0, 1.7, 3.9 and 9.4 mg/L for the test groups containing 10, 22, 46 and 100% WSF, respectively. Growth rate and yield were significantly inhibited at a concentration of 1.7 mg/L (TWA) and higher. Under the study conditions, the 72 h ErC50 for growth rate was > 9.4 mg/L, beyond the range tested and therefore above the maximum solubility of substance in test medium under the study conditions. The 72 h ErC10 was 2.3 mg/L (CI: 1.3 – 4.0 mg/L). The 72 h EbC50 was equivalent to 3.4 mg/L (CI: 2.1 - 5.3 mg/L) and the NOEC for both growth rate and yield was 1.0 mg/L (Migchielsen, 2010).