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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:
2017-08-30 to 2017-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Product name: Vinylcyclohexane
Chemical name: Ethenylcyclohexane
Batch/Lot number: VCH/7/17K1
Purity: 99.8 %
Stability: Stable
Water solubility: 15 mg/L (Analysis Report N0. 3612/17-e, 01.09.2017)
Appearance: Liquid
Colour: Colourless
Storage conditions: Keep containere tightly closed. Keep in a cool, well-ventilated place. Keep away from ignition source.
Expiration date: 30 March 2019
Analytical monitoring:
yes
Details on sampling:
- Concentrations (mg/L): 0 (NC), 12.5 (A), 31.25 (B), 78.13 (C), 195.3 (D), 488.3 (E)
- Sampling method: Chemical analysis of the test item concentrations in the test solutions was performed to determine exposure concentration and stability of the test item during exposure. Substance-specific analysis was performed at the test site Analytisches Zentrum Biopharm GmbH. The samples were filled into 5 mL glass vials and stored in the fridge (5 °C ± 3 °C). The sampling was conducted according to the following specification:
> After 0 h, 24 h and 48 h exposure, both additional vessels of NC, A, B, C, D and E were sampled; one sample per replicate: 2 samples of 5 mL per treatment group.
> After 72 h exposure, both additional vessels and 2 replicates from the test vessels of NC, A, B, C, D and E were sampled; one sample per replicate: 4 samples of 5 mL per treatment group.
Of each sampled treatment, one of the analytic samples from O h, 24 h, 48 h and 72 h was sent to the analytical laboratory at the test site Analytisches Zentrum Biopharm GmbH for chemical analysis, using overnight shipping with an insulated box and thermal packs. The remaining samples were stored as retain samples in the fridge (5 °C ± 3 °C) until finalization of the study.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared as Water Soluble Fraction (WSF) by adding 542.8 mg test item (including a factor of 1.11 to take into account the dilution caused by addition of the algal inoculum) to 1000 mL test medium and shaking for 47.5 h using an overhead shaker at 24.8 — 25.3 °C. The WSF was filtered through a fibre glass filter with a retaining range till 0.6 µm (MN 85/70 BF, Marchery-Nagel, Düren, Germany). The filter was prepared by rinsing with ca. 100 mL purified water and preconditioning with ca. 100 mL WSF (which was discarded), to reduce adsorption of the test item. This filtered stock solution was used as highest test item loading rate in the test. The negative control (NC; test medium) was treated in the same way as the test item solution. Into each test vessel, 50 mL test solution (including algal inoculum) was transferred. The test item loading rates were prepared by diluting the stock solution with test medium according to the following scheme (dilution factor: 2.5).
- Controls: The negative control (NC; test medium) was treated in the same way as the test item solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: /; Scientific name: Raphidocelis subcapitata
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Culture Collection of Algae at the University of Goettingen and was cultivated in suspension culture. The strain used for this study has been cultured at Hydrotox GmbH since May 2016. Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany, Lot no.: MKBF2111V, expiration date: January 2018). The recent quality testing was performed in August 2017 which resulted in an ErC50 of 1.11 mg/L (CL 95 %: 0.65 - 1.86 mg/L; interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 of 0.07 mg/L (CL 95 %: 0.00 – 2.47 mg/L).
- Pre-culture and inoculum: Before the start of the test, 1.19 mL of the algae stock suspension was diluted with 48.81 mL test medium to obtain a concentration of 5 × 10^4 cells/mL. This pre-culture was incubated for 4 d at 22.9 – 23.1 °C and 71.1 µE m-² s-1 ± 3.8 % continuous lighting. For the start of the test, the cell concentration was determined using the Coulter Counter, resulting in 87.401 × 105 cells/mL. To obtain a nominal inoculum concentration of 7 × 10^4 cells/mL, 2.000 mL pre-culture was diluted into 250 mL test medium.

ACCLIMATION
- Acclimation period: 4 d
- Culturing media and conditions (same as test or not): Holm-Hansen Medium
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no
Test temperature:
22.9 – 23.3 °C
pH:
7.6 – 8.1 in the control and 7.7 – 8.0 in the test item treatment
Nominal and measured concentrations:
0, 12.5, 31.25, 78.13, 195.3, 488.3 mg/L (nominal)
0, 28.16, 35.31, 45.85, 55.71, 65.60 µg/L (measured, geometric mean)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer glass flasks 100 mL, Schott, Mainz
- Type (delete if not applicable): open (sealed only with cellulose stoppers)
- Exposure conditions: The test vessels are sealed with cellulose stoppers and kept in the light incubator (specified temperature: 21 – 24 °C, constant within ± 2 °C). They are clipped to rotary discs, with space for 21 vessels on each level. The rotation of ca. 2 rpm ensured that each test vessel is exposed to identical light conditions. Each rotation includes 5 sudden stops by which the test suspensions are shaken. The test vessels were continuously illuminated by lateral fluorescent tubes (58 W each), which were separated from the incubation chamber by heat absorbing glass. Mean light intensity was 71.1 µE m-² s-1 ± 3.8 %. The pH was measured at the start (0 h) and at the end (72 h) of the test.
- Initial cells density: 7 cells x 10³/mL
- Control end cells density: Mean cell count = 3.724 cells x 105/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Holm-Hansen medium

TEST MEDIUM / WATER PARAMETERS
- OECD TG 201 medium according to OECD 201 (2006)

OTHER TEST CONDITIONS
- Sterile test conditions: culturing: yes / test: not reported
- Adjustment of pH: no
- Photoperiod: The test vessels were continuously illuminated by lateral fluorescent tubes (58 W each), which were separated from the incubation chamber by heat absorbing glass. Mean light intensity was 71.1 µE m-² s-1 ± 3.8 %.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: To convert the measured surrogate parameter chlorophyll fluorescence into cell concentration (the measure for biomass), a correlation factor is determined twice a year within the quality check by measuring cell concentrations of different dilutions with the Coulter Counter and the corresponding fluorescence with the microplate fluorescence reader. Chlorophyll fluorescence values are correlated with measured cell concentrations. The slope of the curve gives the conversion factor. By means of the conversion factor, the surrogate parameter chlorophyll fluorescence is converted into cell concentration.
- Chlorophyll measurement: The algal biomass was monitored by measuring the chlorophyll fluorescence in each test vessel after 0 h, 24 h, 48 h and 72 h. From each test vessel, 200 µL test solution was transferred into a 96-well microplate and the fluorescence measured with the fluorescence microplate reader at an excitation wavelength of 465 nm and an emission wavelength of 670 nm. Each measurement was conducted in duplicate. If the variation coefficient was > 10 %, the measurement was repeated. Test medium was measured as blank value and subtracted from the fluorescence values in the test vessels.
- Other: Inspection of cell morphology: At the end of the test, the appearance of the algae was examined microscopically. Any noticeable deviations were documented.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: A preliminary test without GLP was performed before start of this GLP-study. Nominal loading rates of 1, 10 and 100 mg/L test item were tested and resulted in inhibition of -0.3, -25.3 and 24.0 % for yield and -0.6 %, -4.2 % and 5.7 % for growth rate, respectively.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
49 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL = 48 - 51 µg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
39 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL = 38 - 40 µg/L
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
46 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
35 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
45 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
43 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 66 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 66 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algalno test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: yes, in the higher test item loading rates (treatment group C, D and E), some algal cells were thickened and agglutinated.
- Other: /
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: the test item is volatile, hence the differences between measured and nominal concentrations
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate (Sigma, Steinheim, Germany, Lot no.: MKBF2111V, expiration date: January 2018). The recent quality testing was performed in August 2017 which resulted in an ErC50 of 1.11 mg/L (CL 95 %: 0.65 - 1.86 mg/L; interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 of 0.07 mg/L (CL 95 %: 0.00 – 2.47 mg/L).
Reported statistics and error estimates:
With the statistical software ToxRat 3.2.1 (ToxRat Solutions LOEC/NOEC for yield and growth rate were determined.

The test is valid according to OECD Test Guideline 201 (23 March 2006). In the control, the test item was not detected. The initial measured test item concentrations in the test item treatments were 3.79 – 5.66 % (0 h) of the nominal loading rates. Until the end of the exposure period, the measured test item concentrations decreased to < LOQ.

Algae cell concentration

The algae cell concentrations in the control (NC) and the test item treatments are presented in Figure 1.

Cell morphology

The morphology of the algal cells in the lower test item loading rates (treatment group A, B and C) as well as in the control showed no obvious abnormality. In the higher test item loading rates (treatment group C, D and E), some algal cells were thickened and agglutinated.

Yield

The inhibition of the yield in the test item treatment, in relation to the control, is presented in Table 1.

Table 1: Inhibition of yield after 24 h, 48 h and 72 h exposure

Nominal test item loading rate [mg/L]

Inhibition of Yield [%]

24 h

48 h

72 h

12.5

29.9

-6.7

-62.5

31.25

14.0

2.9

-66.3

78.13

38.4

74.5

79.4

195.3

48.8

78.4

94.3

488.3

115.3

100.2

99.9

NC

--

--

--

The inhibition of the specific growth rate in the test item treatment, in relation to the control, is presented in Table 2.

Table 2: Inhibition of growth rate after 24 h, 48 h and 72 h exposure

Nominal test item loading rate [mg/L]

Inhibition of Growth rate [%]

24 h

48 h

72 h

12.5

19.4

-2.0

-11.1

31.25

8.1

0.8

-12.7

78.13

25.4

43.0

38.0

195.3

33.7

47.8

65.3

488.3

141.3

101.4

99.0

NC

--

--

--

Validity criteria fulfilled:
yes
Conclusions:
The 72-hour EC50 (growth rate) for Raphidocelis subcapitata towards vinylcyclohexane was determined with 49 µg/L.
Executive summary:

In a 72-hour acute toxicity study, the cultures of Raphidocelis subcapitata were exposed to vinylcyclohexane at nominal concentrations of 0, 12.5, 31.25, 78.13, 195.3, 488.3 mg/L under static conditions in accordance with the OECD test guideline 201. The NOEC and EC50 values based on growth rate were 35 and 49 µg a.i./L, respectively. 

The following abnormalities were noted: In the higher test item loading rates (treatment group C, D and E), some algal cells were thickened and agglutinated.

This toxicity study is classified as acceptable and satisfies the guideline requirements for freshwater alga and cyanobacteria, Growth Inhibition Tests.

Based on the results of this study, vinylcyclohexane would be classified as toxic to aquatic algae in accordance with the EU CLP Regulation. 

This study is classified as acceptable and satisfies the guideline requirements for a growth inhibition study with aquatic algae.

 

Results Synopsis

Test Organism: Raphidocelis subcapitata

Test Type (Flowthrough, Static, Static Renewal): Static

 

72 hr ErC50: 49 µg a.i./L                95% C.I.:  48 to 51 µg a.i./L

72 hr ErC10: 39 µg a.i./L                95% C.I.:  38 to 40 µg a.i./L

72 hr LOErC: 46 µg a.i./L

72 hr NOErC:  35 µg a.i./L                   

Endpoint(s) Effected: Growth rate

Description of key information

A 72-h static toxicity study with Raphidocelis subcapitata was conducted according to the OECD test guideline 201 and in accordance with the principles of GLP. The derived 72 h EC50 (growth rate) was 49 µg/L based on the geometric mean of measured concentrations. The 72-hr NOEC was 35 µg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:
49 µg/L
EC10 or NOEC for freshwater algae:
35 µg/L

Additional information