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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:
30 Mar - 02 Apr 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
undissolved test material was visible during the test.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: MHLW, (Japan) 13/11/2003, No. 2. Algal Growth Inhibition Test
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations were measured at test start, after 24 and 48 h and at test end.
- Sampling method: At test start, the solutions in the preparation containers were sampled. At 24 and 48 h, the samples were taken from each test vessel. At the end of exposure, samples of the replicates of each test level were mixed and used for measurement.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test sample and medium were mixed and stirred by magnetic stirrer for about 48 h to prepare a nominal concentration of 100 mg/L. After the mixture was centrifuged (10,000 x g, 30 min), the middle layer was collected for the stock solution. Then, the required volume of the stock solution and medium were mixed and stirred in the preparation vessel to prepare the test solution. The test solution was divided into each test vessel.
- Evidence of undissolved material : In the 0.40 and 0.18 mg/L exposure levels, the sticking of white substance was observed on the inner wall of the test vessels around the surface of the test solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: ATCC 22662
- Source: Laboratory culture, originally obtained from the American Type Culture Collection (Rockville MD, USA) on 30 Jun 1995
- Age of inoculum: A pre-culture incubated for 3 days under the same conditions as the test was used as inoculum
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not stated in report
Test temperature:
22.2 - 22.8 °C
pH:
7.7 - 7.9
Dissolved oxygen:
Not stated in report
Nominal and measured concentrations:
Nominal: 0, 1.0, 3.2, 10, 32, 100% stock solution content
Measured: < LOD, 0.0067, 0.021, 0.067, 0.18, 0.40 mg/L (geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: closed with gas-permeable silicon rubber plug
- Material, size, fill volume: glass, 500 mL, 100 mL
- Aeration: none, shaking at 100 rpm
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, as recommended in the OECD 201 guideline

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: Temperature was measured daily, pH and appearance of the test solutions were measured at test start and test end.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 92 - 98 µE/m2*s, fluorescent light with 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Chlorophyll measurement: Chlorophyll fluorescence was measured with a spectrophotofluorometer at 24, 48 and 72 h after test start.
- Other: Condition of the cells was observed for one vessel of each exposure level at test end.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: 0.010, 0.10, 1.0, 10, 100 % stock solution
- Results used to determine the conditions for the definitive study: In the order of the test concentrations, the 72 h growth inhibition rate based on growth rate was -2.6, 0.83, -2.8, 0.26 and 18%, respectively. The measured concentrations of test item were 0.31 mg/L at test start and < LOD at test end for the 10% stock solution nominal level. For 100% stock solution, the measured values were 3.4 mg/L at test start and 0.68 mg/L at test end.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: centrifuged test solution
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.021 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: centrifuged test solution
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes, increase by a factor of 41
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The sticking of white substance on the inner wall of the test vessel around the surface of the test solution was observed in the 0.40 and 0.18 mg/L exposure levels.
- Colour differences: At the start of exposure, test solutions of exposure levels were colourless and clear. At the end of exposure, the appearance of the test solutions in the 0.40 and 0.18 mg/L exposure levels were slightly green and the other exposure levels were green due to algae growth.
- Analytical measurements: The measured concentrations of the test item in the test solutions are given in Table 1. For the two lowest concentrations, reference values calculated from 0.067 mg/L according to the dilution factors were used as mean measured concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- Test date: 06 Jan - 09 Jan 2009
- ErC50 (72 h): 0.8 mg/L (range of laboratory data: 0.53 - 1.4 mg/L)
Reported statistics and error estimates:
Regarding the growth rate, Bartlett's test was done to determine the homogeneity of variance for the data. One-way analysis of variance and Dunnett's multiple comparison test were then done to determine the significant difference between the control level and exposure levels. The statistical analysis was conducted using a computer program running on MS Excel developed in the laboratory. The NOEC was determined by the results of statistical analysis and cell condition.

Table 1: Measured concentrations of the test item in the test solutions

Stock solution (%)

Measured concentration (mg/L)

0 h

24 h

48 h

72 h

Geometric mean

Control

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

-

1.0

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

-

3.2

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

-

10

0.18

< 0.11 mg/L

< 0.11 mg/L

< 0.11 mg/L

0.067

32

0.58

0.24

0.14

< 0.11 mg/L

0.18

100

1.9

0.49

0.21

0.22

0.40

Table 2: Measurement of Chlorophyll fluorescence

Measured concentration (mg/L)

Chlorophyll fluorescence (relative unit)

0 h

24 h

48 h

72 h

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Control

3.9

0

16

1.2

69

7.4

170

10

0.0067

3.9

0

16

0.52

71

2.5

190

13

0.021

3.9

0

14

0.74

61

4.4

160

1.6

0.067

3.9

0

14

1.5

58

6.4

150

6.6

0.18

3.9

0

13

0.81

51

2.1

120

5.3

0.40

3.9

0

13

0.49

46

1.4

95

4.0

Table 3: Growth rates and growth inhibition, ** significant difference to control (p < 0.01)

Measured concentration (mg/L)

Growth rate 72 h

Inhibition rate (%)

Mean

SD

Mean

SD

Control

1.3

0.020

-

-

0.0067

1.3

0.024

-2.4

1.9

0.021

1.2

0.033

2.6

0.26

0.067

1.2

0.015

3.8**

1.2

0.18

1.1

0.015

10**

1.2

0.40

1.1

0.014

16**

1.1

Validity criteria fulfilled:
yes
Conclusions:
ErC50 of the test material based on growth rate was > 0.40 mg/L (95% confidence intervals not calculated).
Executive summary:

The study was performed according to the following test methods and guidance documents.

1) Algal Growth Inhibition Test stipulated in the "Testing Methods for New chemical substances" (November 21, 2003; No. 1121002, Pharmaceutical and Food safety bureau, MHLW; November 13 2003, No. 2, Manufacturing Industries Bureau, METI; No. 031121002, Environmental Policy Bureau, Ministry of the Environment, Partial amendment November 20, 2006.

2) OECD guidelines for Testing of Chemicals, Section 2: Effects of Biotic systems, "Freshwater Algae and Cyanobacteria; Growth Inhibition Test (Guideline 201, March 23, 2006).

3) OECD Guidance Document 23 "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures" (September 2000).

Five exposure levels of 100, 32, 10, 3.2 and 1.0% (a geometric series with a factor of √10) as stock solution content [0.40, 0.18, 0.067, 0.021 (reference value) and 0.0067 (reference value) mg/L as measured concentration] and a control.

Concentration of test item in test solution '

[Percent of measured concentration versus that at the start the exposure]

At start of the exposure: Below the determination limit (<0.11 mg/L) to 1.9 mg/L.

After 24 hours

Below the determination limit (<0.11 mg/L) to 0.49 mg/L.

[25% (in the stock solution content 100% test level)]

After 48 hours

Below the determination limit (<0.11 mg/L) to 0.21 mg/L

[11 % (in the stock solution content 100% test level)]

At the end of the exposure

Below the determination limit (<0.11 mg/L) to 0.22 mg/L

[11 % (in the stock solution content 100% level)]

ErC50 > 0.40 mg/L (95% confidence limit not calculated)

NOEC (Growth rate - 3 -days) 0.021 mg/L

(ErC50 and NOEC based on geometric mean of measured concentrations)

Description of key information

ErC50 (72 h) > 0.4 mg/L and NOEC = 0.021 mg/L for Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
0.4 mg/L
EC10, LC10 or NOEC for freshwater algae:
0.021 mg/L

Additional information

For the toxicity of the test item to aquatic algae one key study with Pseudokirchneriella subcapitata according to OECD 201 and GLP is available (Showa Denko, 2009). The algae were exposed to analytically verified concentrations of the test item under static conditions. After 72 h of exposure, the growth inhibition at the highest concentration tested (0.4 mg/L mean measured concentration) was 16%. Therefore, the ErC50 (72 h) was determined to be > 0.4 mg/L and the NOEC was established as 0.021 mg/L. The concentration given for the NOEC is a reference value calculated from the content rate of stock solution as the two lowest nominal test levels were below the limit of quantification.