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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:
18th, January 2014 - 21st January, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
no
Remarks:
GLP not required at time of test initiation
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Maplewood, Lot# 20125
- Expiration date of the lot/batch: >1 year after manufacture date (29th October, 2013)
- Purity test date: 14th January, 2014
- Appearence: Liquid, brown-green

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature (15 to 30 °C)
- Stability under test conditions: Stable at room temperature. Can oxidize in presence of oxygen at high temperatures.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAFs were prepared at concentrations of 0, 0.07, 0.14, 0.28, 0.56 and 1.12 mg/L, applying a 48 hour period of stirring with a stainless steel propeller (250 rpm) to attempt dissolution of the test item in medium. Vessels were wrapped in aluminum foil during stirring to exclude light. The test item was allowed to settle for one hour, and then the test solution was siphoned from the middle of the glass (first ca. 100 mL discarded).
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material: At the end of the WAF preparation/test initiation, all test solutions were clear and colorless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CPCC 37, CP131009
- Source: Canadian Phycological Culture Center, Waterloo, ON, Canada
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: propagation under same conditions as the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23-25 °C
pH:
Starting pH:7.3-7.5
Ending pH:7.6-7.8
Conductivity:
88-95 µS/cm
Nominal and measured concentrations:
Nominal concentrations: 0 (WAF blank control), 0.07, 0.14, 0.28, 0.56, and 1.12 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer glass flasks
- Type: Closed
- Aeration: No
- Initial cells density:10,000 cells/mL
- Control end cells density: 2,080,000 cells/mL (lab control) and 1,460,000 cells/mL (WAF control)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6. Two controls were used (WAF and lab water)

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used: NaNO3 (25500 ug/L), MgCl2 6H20 (10,000 ug/L), CaCl2 2H2O (4420 ug/L), H3BO3 (186 ug/L), MnCl2 4H2O (416 ug/L), ZnCl2 (3.28 ug/L), COCl2 6H2O (1.43 ug/L), CuCl2 2H2O (0.012 ug/L), Na2MoO4 2H2O (7.27 ug/L), FeCl3 6H2O (160 ug/L), Na2EDTA 2H20 (300 ug/L), MgSO4 7H2O (14700 ug/L), K2HPO4 (1044 ug/L), NaHCO3 (15000 ug/L).

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 hour
- Light intensity and quality: 6152-7662 lux. No information available in study report on light quality.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations: Cellometer AutoX10 counter at 24, 48 and 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2X
- Range finding study: Yes
- Test concentrations: 0, 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. 72 hour IC50=0.18 mg/L
Reference substance (positive control):
yes
Remarks:
Zinc sulphate heptahydrate (CAS# 7446-20-0)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
0.43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.40-0.47 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): Yes
- Growth rates observed for the WAF control and medium control
- Observation of abnormalities: No observations made
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- EC50: 0.094 mg Zn/L
- A 72 hour acute toxicity test of zinc sulphate heptahydrate was done as part of the positive control testing around the time of this test. The IC50 was 0.094 mg/L. This was within the control range, where range is twice the standard deviation.
- Reference test was run 7 days after the definitve test.
Reported statistics and error estimates:
Nonlinear regression was used to determine the IL50/25 for specific growth rate and cell yield. THe statistical program CETIS (V 1.7.0.3) was used to calculate IL50/25 and their 95% confidence intervals in addtiion to the NOELR/LOELR values. The CETIS package ran a Shapiro-Wilk Normality test for normal distribution, a Mann-Kendall Trend to examine for significant trends in controls, a Grubbs Extreme Value test to look for outliers, a modified Levene Equality of Vaeience to determine euqal variances, and a Dunnetts Multiple Comparison Test on the stnadard groth rate and cell yield endpoints.

Table 1, Cell counts  (x 104cells/mL) during the algae toxicity test

Mean concentration (mg/L)

T = 0 hour

T = 24 hour

T = 48 hour

T = 72 hour

Control

1.0

4.1

23.9

202.5

 

1.0

4.1

34.3

242.3

 

1.0

8.4

25.6

226.0

 

1.0

3.8

26.5

218.0

 

1.0

3.2

28.4

190.8

 

1.0

3.2

24.5

166.8

Mean

 

 

 

207.7

WAF Control

1.0

5.0

31.1

198.5

 

1.0

4.1

24.3

123.8

 

1.0

3.5

17.9

113.7

 

1.0

4.9

22.0

84.6

 

1.0

5.5

32.5

164.0

1.0

3.6

52.3

190.5

Mean

 

 

 

145.8

0.07

1.0

3.0

23.2

137.0

 

1.0

4.7

16.0

120.0

 

1.0

6.2

27.9

153.0

Mean

 

 

 

136.7

0.14

1.0

1.5

14.4

102.8

 

1.0

2.6

16.9

90.1

 

1.0

3.6

21.5

164.0

Mean

 

 

 

118.9

0.28

1.0

1.5

12.1

88.9

 

1.0

0.6

5.6

63.5

 

1.0

1.0

2.7

77.9

Mean

 

 

 

76.8

0.56

1.0

0.0

0.3

4.4

 

1.0

0.0

0.3

1.4

 

1.0

0.0

0.0

1.4

Mean

 

 

 

2.4

1.12

1.0

0.0

0.0

0.0

 

1.0

0.0

0.0

0.0

 

1.0

0.0

0.0

0.0

Mean

 

 

 

0.0

 

Table 2. Growth rates (1/day) during the algae toxicity test.

Mean concentration (mg/L)

T = 0-24 hour

T = 24-48 hour

T = 48-72 hour

T = 0-72 hour

Control

1.42

1.76

2.14

1.77

 

1.41

2.13

1.96

1.83

 

2.13

1.11

2.18

1.81

 

1.33

1.95

2.11

1.80

 

1.18

2.17

1.91

1.75

 

1.17

2.04

1.92

1.71

Mean

1.44

1.85

2.03

1.78

SD

0.36

0.39

0.12

0.0445

WAF Control

1.62

1.82

1.86

1.76

 

1.41

1.78

1.63

1.61

 

1.26

1.62

1.85

1.58

 

1.60

1.49

1.35

1.48

 

1.71

1.77

1.62

1.70

1.28

2.68

1.29

1.75

Mean

1.48

1.86

1.60

1.65

0.07

1.10

2.05

1.78

1.64

 

1.55

1.23

2.02

1.60

 

1.82

1.51

1.70

1.68

Mean

1.49

1.60

1.83

1.64

0.14

0.40

2.27

1.97

1.54

 

0.95

1.88

1.68

1.50

 

1.27

1.80

2.03

1.70

Mean

0.87

1.98

1.89

1.58

0.28

0.38

2.12

2.00

1.50

 

-0.55

2.27

2.43

1.38

 

-0.04

1.03

3.37

1.45

Mean

-0.07

1.81

2,60

1.44

0.56

N/C

N/C

2.66

0.5

 

N/C

N/C

1.49

0.12

 

N/C

N/C

N/C

0.12

Mean

N/A

N/A

2.08

0.25

1.12

N/C

N/C

N/C

N/C

 

N/C

N/C

N/C

N/C

 

N/C

N/C

N/C

N/C

Mean

N/A

N/A

N/A

N/A

%CV of WAF control average growth rate is 6.6

%CV of WAF control section-by-section growth rate is 16%

%CV of Blank control section-by-section growth rate is 26%

N/C- Not Calculable, N/A- Not Available

Validity criteria fulfilled:
yes
Remarks:
control biomass increase >16x (208x), CV for section-by-section g rowth rates <35% (26%), CV for average specific growth rates <7% (2.50%)
Conclusions:
72 hour EL50 = 0.43 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
72 hour NOELR = 0.28 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).
Executive summary:

The 72 hour EL50 to Pseudokichneriella subcapitata was determined in a definitive test according to OECD 201 guidelines. Nominal concentrations of 0.07, 0.14, 0.28, 0.56 and 1.12 mg/L, and a WAF control were run using 3 replicates per concentration (6 replicates for water and WAF controls) and no dispersant. An EL50 of 0.43 mg/L and a NOELR of 0.28 mg/L were determined. This study was conducted under international guidelines but is not GLP compliant. In addition, no analytical monitoring was conducted. Therefore, this study is considered reliable with restrictions. The result is considered suitable for use in Risk Assessment, Classification & Labeling, and PBT analysis.

Description of key information

72 hour EL50 = 0.43 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).

72 hour NOELR = 0.28 mg/L (nominal) in Pseudokichneriella subcapitata (OECD 201).

Key value for chemical safety assessment

EC50 for freshwater algae:
0.43 mg/L
EC10 or NOEC for freshwater algae:
0.28 mg/L

Additional information