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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 March 2012 - 02 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
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
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 7831
- Substance type: Pure active substance
- Physical state: Waxy solid
- Analytical purity: 93.96 w%
- Expiration date of the lot/batch: 31 October 2016
- Stability under test conditions: Yes
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations monitored
- Sampling method: One 2-mL sample was taken from all test concentrations and the control at times zero, 24h and 72h. One sample was held in reserve.
- Sample storage conditions before analysis: Stored frozen (≤ 15 °C)
- Abiotic samples: To verify maintanence of test substance concentration, a test vessel was set up at the 5.0 mg/L loading rate without algae. Samples for analysis were taken at times zero, 24h and 72h.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Loading rates of 0.5, 1.6 and 5.0 mg/l were prepared by 30 minutes of ultrasonic treatment followed by 1 day of magnetic stirring. The resulting dispersions were left to settle for a maximum of 30 minutes after which the clear Water Accommodated Fractions (WAFs) were collected by siphoning. Two lower test concentrations were prepared by dilution of the WAF prepared at 0.5 mg/l. The pH of the test medium was not adjusted.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): No visible undissolved test substance was observed at test initiation. The final test solutions were all clear and colourless.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1E+04 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: None.
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
21.2 - 23.3 °C
pH:
8.0 - 8.2
Nominal and measured concentrations:
Nominal concentrations (loading rate): 0.05 mg/L, 0.16 mg/L, 0.5 mg/L, 1.6 mg/L, 5.0 mg/L
Time Weighted Average measured concentrations: 0.02 mg/L, 0.02 mg/L, 0.32 mg/L, 0.074 mg/L, 0.34 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, and capped. Fill volume was 50 mL
- Agitation: Yes, during incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 70.2E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
1 replicate of each test concentrations without algae
1 or 2 extra replicates of each test group with algae for sampling after 24 hours.

GROWTH MEDIUM
- Standard medium used: Yes - M1 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis)
- Culture medium different from test medium: yes, propagation in M1, test in adjusted M2 but a pre-culture established in M2 medium 3 days before test initiation to use in test
- Intervals of water quality measurement: pH at beginning and 72 hours, temperature continuously

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted in the range-finding test but not the final test
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 90 to 94 mE/(m²∙s).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Beginning of the test, cells were counted with a microscope and counting chamber. Thereafter cell densities were measured using a spectrophotometer with immersion probe (pathlength = 20 mm). At the end of the study final microscopic observations were made on the highest test concentration to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 (0.5 log unit)
- Range finding study: Yes
- Test concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/l, with an additional ten-fold dilution prepared from the solution obtained from the 1.0 mg/L loading.
- Results used to determine the conditions for the definitive study: Yes. The expected EC50 for growth rate exceeded the test range (exceeded the limit of solubility in the test medium).
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.34 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 72 h NOEC (growth rate): 0.34 mg/L. The ErC50 and NOEC results correspond to nominally 5.0 mg/L C4 acrylate.
Details on results:
- Exponential growth in the control (for algal test): yes
- Growth in controls (validity criteria):
- cell density increase within 72 hours: factor of >16
- % Coefficient of variance in average specific growth rate at hour 72: 2% (Table 1)
- Mean % coefficient of variance in section-specific growth rates to hour 72: 6% (Table 2)
The measured concentrations are in table 3 and the effect parameters are in table 4.

- Observation of abnormalities (for algal test): Observation was done at the end of the test and no abnormalities were found.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: C4 Acrylate is not completely soluble in the test medium. The measured concentrations for the two dilutions prepared from the 0.5 mg/L WAF were below the level of detection (LOD: <0.04 mg/L). Measured concentrations of the WAFs decreased significantly after 24 hours and all concentrations were LOD (< 0.04 mg/L) after 72 hours of exposure. The highest test concentration (nominal: 5mg/L) incubated without algae showed a similar decrease. Time Weighted Average (TWA) exposure concentrations were calcualted for the three highest WAFs (0.5, 1.6, and 5.0 mg/L).
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50: 1.7 mg/L, 95% C.I. 1.2 - 2.3 mg/L. The historical ranges for growth rate reduction for Pseudokirchneriella subcapitata lie between 0.82 and 2.3 mg/l. Hence, the growth rate reduction EC50: 72h for the Pseudokirchneriella subcapitata tested corresponds with this range.

Any other information on results incl. tables

Fig 1 : growth curves.jpg ---> see attached background material

Table 1: Cell Density, Yield Inhibition and Growth Rate Reductions

Nominal WAF

Concentration

C4 Acrylate

[mg/L]

Vessel Number

Individual Cell Densities, 104 cells/mL

Yield

Yield Inhibition

(%)

Growth Rate (µ)

Growth Rate (µ) Reduction (%)

Exposure Period (hours)

0

24

48

72

0 - 72 h

0 - 72 h

0 - 72 h

0 - 72 h

Control

1

1.00

3.67

14.27

68.56

67.56

0.05872

2

1.00

3.87

16.52

79.94

78.94

0.06085

3

1.00

4.26

18.26

75.99

74.99

0.06015

4

1.00

3.70

14.26

61.91

60.91

0.05730

5

1.00

4.13

16.57

66.48

65.48

0.05829

6

1.00

3.78

14.91

68.14

67.14

0.05863

Mean

69.17

0.05899

CV 2%

0.05*

(0.020)

1

1.00

4.16

15.54

73.18

72.18

-4

0.05962

-1

2

1.00

4.41

17.31

81.35

80.35

-16

0.06109

-4

3

1.00

4.31

15.23

74.56

73.56

-6

0.05988

-2

Mean

75.36

-9

0.06020

-2.1

0.16**

(0.020)

1

1.00

4.21

17.97

77.64

76.64

-11

0.06045

-2

2

1.00

4.28

15.07

65.88

64.88

6

0.05816

1

3

1.00

4.72

15.66

65.84

64.84

6

0.05816

1

Mean

68.79

0.6

0.05892

0.1

0.5

(0.032)

1

1.00

4.20

14.32

60.40

59.40

14

0.05696

3

2

1.00

4.41

16.11

75.38

74.38

-8

0.06004

-2

3

1.00

4.35

14.35

72.89

71.89

-4

0.05957

-1

Mean

68.56

0.9

0.05885

0.2

1.6

(0.074)

1

1.00

4.22

16.54

70.34

69.34

0

0.05907

0

2

1.00

4.51

17.70

73.45

72.45

-5

0.05967

-1

3

1.00

4.43

15.61

68.62

67.62

2

0.05873

0

Mean

69.80

-0.9

0.05916

-0.3

5.0

(0.34)

1

1.00

4.15

16.04

64.75

63.75

8

0.05792

2

2

1.00

4.14

15.07

68.32

67.32

3

0.05867

1

3

1.00

4.27

14.38

56.63

55.63

20

0.05606

5

Mean

62.23

10.0

0.05755

2.4

* 10% of the WAF prepared at 0.5 mg/l, ** 32% of the WAF prepared at 0.5 mg/l

( ) Between brackets: TWA concentration.

Table 2: Section-by-section Growth Rates

Nominal WAF

Concentration

C4 Acrylate

[mg/L]

Vessel Number

Growth Rate (µ)

Growth Rate Reduction (%)

Exposure Period (hours)

0 - 24h

24-48h

48-72h

0 - 24h

24-48h

48-72h

Control

1

0.05414

0.05662

0.06539

2

0.05641

0.06046

0.06569

3

0.06036

0.06068

0.05941

4

0.05455

0.05618

0.06118

5

0.05913

0.05786

0.05789

6

0.05545

0.05713

0.06332

Mean

0.05667

0.05815

0.06215

CV (%)

4

3

5

The mean CV for section-by-section specific growth rate was: 6%

0.05*

(0.020)

1

0.05935

0.05495

0.06458

-5

6

-4

2

0.06180

0.05699

0.06449

-9

2

-4

3

0.06083

0.05262

0.06619

-7

10

-7

Mean

0.06066

0.05485

0.06509

-7

5.7

-4.7

0.16**

(0.020)

1

0.05985

0.06051

0.06097

-6

-4

2

2

0.06053

0.05249

0.06147

-7

10

1

3

0.06467

0.04997

0.05983

-14

14

4

Mean

0.06168

0.05432

0.06076

-8.8

6.6

2.2

0.5

(0.032)

1

0.05981

0.05109

0.05998

-6

12

3

2

0.06180

0.05401

0.06430

-9

7

-3

3

0.06125

0.04974

0.06772

-8

14

-9

Mean

0.06095

0.05161

0.06400

-7.6

11.2

-3.0

1.6

(0.074)

1

0.05997

0.05692

0.06033

-6

2

3

2

0.06273

0.05700

0.05930

-11

2

5

3

0.06197

0.05253

0.06169

-9

10

1

Mean

0.06156

0.05548

0.06044

-8.6

4.6

2.7

5

(0.34)

1

0.05928

0.05635

0.05815

-5

3

6

2

0.05915

0.05387

0.06299

-4

7

-1

3

0.06051

0.05057

0.05711

-7

13

8

Mean

0.05964

0.05360

0.05942

-5.2

7.8

4.4

* 10% of the WAF prepared at 0.5 mg/l, ** 32% of the WAF prepared at 0.5 mg/l
( ) Between brackets: Measured TWA concentration.

Table 3. Measured concentrations in the final test.

Time of

sampling

[hours]

 

Loading Rate ¹

C4 Acrylate

[mg/l]

Analysed

concentration

C4 Acrylate

[mg/l]

Relative to

initial

[%]

0

0

<LOD

 

10% of 0.5

<LOD

 

32% of 0.5

<LOD

 

0.5

0.158

 

1.6

0.534

 

5.0

1.44

 

5.0 ²

1.49

 

24

0

<LOD

n.a.

10% of 0.5

<LOD

n.a.

32% of 0.5

<LOD

n.a.

0.5

<LOD

< 26

1.6

0.486 ³

9

5.0

0.473

33

5.0 ²

0.728

49

72

0

<LOD

 

10% of 0.5

<LOD

 

32% of 0.5

<LOD

 

0.5

<LOD

< 26

1.6

<LOD

< 8

5.0

<LOD

< 3

5.0 ²

<LOD

< 3

1) A water accommodated fraction (WAF)

2) Without algae

3) Obtained by extrapolation of the calibration curve
LOD – limit of detection (LOD=0.04 mg/l)

n.a. Not applicable

Table 4. Effective concentrations and nominal concentrations of C4 Acrylate

Inhibition

Parameter

Measured TWA

concentration (mg/L)

Nominal concentrations (mg/L)

NOEC

(growth rate)

0.34

5.0

72h EC50

(growth rate)

> 0.34

> 5.0

NOEC

(yield)

0.34

5.0

72h EC50

(yield)

> 0.34

> 5.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Cell density increased within 72 hours by an average factor of >16. Control cultures mean coefficient of variation section-by-section specific growth rates < 35%. Control cultures coefficient of variation of specific growth rates during whole test < 7%.
Conclusions:
The 72-hour ErC50 of C4 acrylate to Pseudokirchneriella subcapitata is > 0.34 mg/L. The 72-hour NOEC of C4 acrylate to Pseudokirchneriella subcapitata is 0.34 mg/L based on growth rate.
Executive summary:

The toxicity of C4 acrylate to the green algae, Pseudokirchneriella subcapitata, was assessed in a 72-hour toxicity test conducted according to the OECD 201 (2011) method. An exponential growth was observed during the entire period of exposure in the control vessel and the validity criteria were met.

The 72-hour ErC50 of C4 acrylate to Pseudokirchneriella subcapitata for growth rate was > 0.34 mg/L being the Time Weighted Average (TWA) concentration measured from a Water Accommodated Fraction (WAF) prepared at 5.0 mg/L.

The study was performed in accordance with internationally-accepted test guidelines and Good Laboratory Practice (GLP) standards. Therefore, this study is reliable without restrictions and the results are suitable for purposes of Risk Assessment, Classification & Labeling, and PBT Analysis.

Results Synopsis

Test Type: static (based on data obtained using OECD 201 methodology)

72-hr ErC50: >0.34 mg/L

72-hr NOEC: 0.34 mg/L

The ErC50 and NOEC results correspond to nominally 5.0 mg/L C4 acrylate.