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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:
2009-09-11 to 2009-10-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 and the EC Method C.3 with GLP certificate. All validity criteria were fulfilled and no deviations were observed. Therefore, this study is considered reliable without restriction.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
1992
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss GLP Monitoring Authority (inspected on November 05-09 and 26-30, 2007/signed on November 12, 2008)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: For measurement of the actual concentrations of the test item, 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 the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. The concentrations of the test item were determined in the duplicate test medium samples from the nominal test concentrations of 10 to 100 mg/L. The samples from the nominal test concentrations of 0.10 to 3.2 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 100.10 mg of the test item completely in 1000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 24 hours at room temperature in the dark. The test medium of the highest test concentration was diluted with test water to prepare the test media. The test media were prepared just before the start of the test.
- Eluate: Test water
- Controls: Yes (test water without test item)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen/Germany).
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
Test temperature:
The test flasks were incubated in a temperature-controlled water bath at a temperature of 22-23 °C
pH:
At the start of the test, the pH measured in the treatments was between 8.0 and 8.2. At the end of the test, pH values of 8.2 to 8.9 were measured.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: Covered with a glass dish
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks
- Aeration: During the test, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: At the start of the test, 10000 cells/mL were inoculated.
- Control end cells density: 118.1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes; OECD TG 201 medium
- Culture medium different from test medium: No

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).
- The pH was measured and recorded in each treatment at the start and at the end of the test. 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. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Light intensity and quality: Approximately 7700 Lux (range: 7170 to 8250 Lux); illuminated by fluorescent tubes

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter
- Other: A small volume of the algal suspension was daily withdrawn from each test flask for the measurement of the biomass, and was not replaced. The algal biomass in the samples was determined by measurement of the algal cell density using an electronic particle counter (Coulter Counter®, Model ZM). The measurements were performed at least in duplicate. At the end of the test, a sample was taken from the control and from the test concentration of nominal 32 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected. This test concentration was chosen since the algal cell density was too low for a reliable examination at the highest nominal concentration of 100 mg/L.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes (non-GLP)
- Results used to determine the conditions for the definitive study: No data
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (Study No. C36893)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 24-28 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 10-14 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 85-90 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 30-35 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
7.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- The test item had a significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the mean measured concentration of 23 mg/L and at the higher test concentration (results of Dunnett’s tests, one-sided, α = 0.05). Thus, this concentration was determined to be the 72-hour LOEC. The 72-hour NOEC was determined to be 7.1 mg/L, since up to and including this test concentration, the growth rate and yield of the algae after 72 hours were not significantly lower than in the control.
- 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 32 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to this concentration.
- In the control the biomass increased by a factor of 118 over 72 hours (Table 1). The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, Table 4) during 72 hours was 12%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 2.6%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.
- No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- At the start of the test, the pH measured in the treatments was between 8.0 and 8.2. At the end of the test, pH values of 8.2 to 8.9 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test.
- The measured concentrations of the test item in the test media of the nominal test concentrations of 10 to 100 mg/L were between 99 and 101% of the nominal values at the start of the test (see analytical results and Table 2 in the corresponding appendix). During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 50 to 86% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 71 and 92% of the nominal values.
Results with reference substance (positive control):
Result of the positive control test performed in February 2009 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.1 mg/L (study C36893), range of the 72-hour EC50 for the growth rate from 2000 to 2009: 0.71-1.74 mg/L).
Reported statistics and error estimates:
- The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis.
- For the determination of the LOEC and NOEC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett’s tests.

Table 6.1.5/1: Average Growth Rates (μ)

Nominal test item concentration

(mg/L)

 

Mean measured concentration

(mg/L)

 

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

 

0-24 h

 

0-48 h

 

0-72 h

 

μ

 

Ir (%)

 

μ

 

Ir (%)

 

μ

 

Ir (%)

 

Control

 

-

1.72

0.0

1.66

0.0

1.59

0.0

0.10

-

1.57*

8.8

1.55*

6.9

1.57

1.1

0.32

-

1.62

5.7

1.63

2.1

1.61

-1.1

1.0

-

1.67

2.5

1.63

1.8

1.59

-0.2

3.2

-

1.57*

8.5

1.66

0.2

1.59

-0.3

10

7.1

1.52*

11.6

1.63

1.8

1.57

1.4

32

23

1.39*

19.0

1.47*

11.7

1.47*

7.6

100

92

1.11*

35.4

0.89*

46.2

0.76*

52.5

*: mean value significantly lower than in the control (according to Dunnett’s-tests, one-sided, α = 0.05)

Table 6.1.5/2: Yield (Y)

Nominal test item concentration

(mg/L)

 

Mean measured concentration

(mg/L)

 

Yield Y (bei Zellzahl: x 104) and inhibition of Y (Iy)

 

0-24 h

 

0-48 h

 

0-72 h

 

Y

Iy (%)

Y

Iy (%)

Y

Iy (%)

Control

-

4.6

0.0

26.8

0.0

117.1

0.0

0.10

-

3.8*

16.9

21.1*

21.5

110.4

5.7

0.32

-

4.1*

11.5

24.9

7.4

123.1

-5.1

1.0

-

4.3

5.3

25.3

5.8

118.3

-1.0

3.2

-

3.8*

16.6

26.5

1.2

118.2

-0.9

10

7.1

3.6*

22.0

25.1

6.5

108.7

7.2

32

23

3.0*

34.1

17.9*

33.5

80.7*

31.1

100

92

2.0*

55.6

5.0*

81.4

8.7*

92.6

*: mean value significantly lower than in the control (according to Dunnett’s-tests, one-sided, α = 0.05)

Analysis of concentrations: The measured concentrations of the test item in the test media of the nominal test concentrations of 10 to 100 mg/L were between 99 and 101% of the nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 50 to 86% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 71 and 92% of the nominal values.

Validity criteria fulfilled:
yes
Conclusions:
Based on geometric mean measured concentrations, the 72h-NOEC (growth rate) and 72h-EC50 (growth rate) were 7.1 mg/L and 87 mg/L, respectively.
Executive summary:

In a algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at the nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions.

The measured concentrations of the test item in the test media of the nominal test concentrations of 10 to 100 mg/L were between 99 and 101% of the nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 50 to 86% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 71 and 92% of the nominal values.

 

The test item had a significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the mean measured concentration of 23 mg/L and at the higher test concentration. The test item had no significant inhibitory effect on the growth of the algae (average growth rate and yield) during the test period of 72 hours at the mean measured concentration of 7.1 mg/L. The 72-h NOEC and LOEC were 7.1 and 23 mg/L, respectively. The 72 -hour EC50 was 87 and 32 mg/L for growth rate and yield, respectively.

Description of key information

OECD Guideline 201, EU Method C.3, GLP, key study, validity 1:
72h-NOEC (growth rate) = 7.1 mg/L (geometric mean measured concentration),
72h-EC50 (growth rate) = 87 mg/L (geometric mean measured concentration).

Key value for chemical safety assessment

EC50 for freshwater algae:
87 mg/L
EC10 or NOEC for freshwater algae:
7.1 mg/L

Additional information

One valid key study is available to assess the toxicity of the substance to aquatic algal. This growth inhibition study was performed according to OECD Guideline 201 and EU method C.3, in compliance with GLP. The freshwater green algae species Pseudokirchneriella subcapitata was exposed to test material at the nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions.

The measured concentrations of the test item in the test media of the nominal test concentrations of 10 to 100 mg/L were between 99 and 101% of the nominal values at the start of the test. During the test period of 72 hours, a decrease of test item concentration in the test media occurred. At the end of the test, 50 to 86% of the nominal values were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) were between 71 and 92% of the nominal values.

The 72-h NOEC and LOEC were 7.1 and 23 mg/L (based on geometric mean measured concentrations), respectively. The 72 -hour EC50 was 87 and 32 mg/L for growth rate and yield ((based on geometric mean measured concentrations), respectively.