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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:
From June 11th, 2012 to December 20th, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: 3 ml were taken from all test concentrations and the control at t=0, t=24h and t=72h.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a concentration of 100 mg/l applying a 30-minute period of magnetic stirring to accelerate the dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.
- Controls:Test medium without test substance or other additives.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Method of cultivation: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (adjusted-M2) at a cell density of 10E+04 cells/ml. The pre-culture was maintained under the same conditions as used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
22.2-23.5°C
pH:
7.1-7.8
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32, 100, 100 (without algae) mg/L
Measured concentration (Time Weight Average): 0 (control), 0.30, 0.96, 4.2, 8.3, 40, 63 (without algae) mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 ml, all-glass
- Type (delete if not applicable): airtight closed with a septum
- Headspace, fill volume: 120 ml of test solution, with no headspace
- Control end cells density: 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3 replicates of each test concentration, 1 or 2 replicated of each test concentration without algae, 1 additional replicate of each test concentration for sampling purposes.
- No. of vessels per control (replicates): 6 replicates.

GROWTH MEDIUM (Stock culture medium)
- Standard medium used: M1 according to the NPR 6505 formulated using Milli-RO water.
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted-M2 medium (according to OECD 201). Based on the preliminary experiment, due to volatile characteristics of the test compound the study was performed without headspace in air-tight closed vessels with a septum. No CO2 from the headspace could diffuse into the test medium and therefore, the medium was adjusted to compensate for this loss with 300 mg/L NaHCO3 and 6 mmol/L HEPES buffer.
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis).
- Culture medium different from test medium:
Stock culture medium: M1 according to the NPR 6505 formulated using Milli-RO water
Pre-culture medium (3 days before the start of the test) and the test medium: adjusted-M2 medium (see above).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 65 to 76 µE.m-2.s-1.
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm) at t=0 h, t=24 h and t=72 h.
- Other: At the end of the final test microscopic observations were performed on the highest concentration to observe for any abnormal appearance of the algae
- Mean cell densities, reduction of growth rate and inhibition of yield.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/l and three replicates per concentration were exposed to 0.1, 1.0 and 10 mg/L test item for 72h in M2 medium (without adjustment and open vessels).
- Test concentrations: 0 (control), 0.1, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: No effects were observed on the growth rate at any of the tested concentrations. In the highest concentration the yield was inhibited by 5.9%. Therefore, the expected EC50 for growth rate reduction and yield inhibition was above a nominal concentration of 100 mg/L.
Reference substance (positive control):
no
Remarks:
(Internal laboratory results of the reference test with potassium dichromate were included)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 40 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
15 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CL: 5.7-38 mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Details on results: Growth rates were in the range of the controls at the TWA concentrations from 0.30 to 4.2 mg/l during the 72-hour test period, whereas the growth rate of algae exposed to 8.3 and 40 mg/l were increasingly reduced (Bonferroni t test, α = 0.05). Inhibition of yield increased with increasing TWA concentration of 2-methylcyclohexylacetate. Statistically significant inhibition of yield was found at TWA concentrations of 8.3 mg/l and higher (Bonferroni t test, α = 0.05).
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
- Analytical monitoring: Despite the fact that vessels were completely filled and airtight closed, the concentrations did not remain stable during the test period (8.8-20% of initial). Consequently, Time Weight Average (TWA) concentrations were calculated to determine the effect levels.
Results with reference substance (positive control):
- Results with reference substance valid? Yes (historical data with potassium dichromate, November 2012)
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.7 mg/l with a 95% confidence interval ranging from 1.2 to 2.3 mg/l.
- Other: The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Calculation of the EC50 and EC10 values was based on log-linear regression analysis of the percentages of growth rate reduction and the percentages of yield inhibition versus the logarithms of the corresponding average exposure of the test substance. No EC50 could be calculated for growth rate as the test substance proved to be not toxic (EC50> than the maximum concentration tested). The EC50 values could only be estimated for yield inhibition (EYC50 (0-72h)). An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant reduction of growth rate or inhibition of yield.

Percentage reduction of growth rate (total test period) and percentage inhibition of yield during the final test:

TWA concentration

2-methylcyclohexyl acetate (mg/l)

Mean growth rate

Yield (0-72 h)

µ (0-72 h)

Reduction (%)

x104cells/ml

Inhibition (%)

control

0.06874

141.03

0.30

0.06947

-1.1

147.97

-4.9

0.96

0.06678

2.9

122.29

13.3

4.2

0.06806

1.0

135.08

4.2

8.3

0.06475

5.8*

105.14

25.4*

40

0.05695

17.2*

59.47

57.8*

Percentage reduction of growth rate at different time intervals during the final test:

TWA concentration

2-methylcyclohexyl acetate (mg/l)

Mean growth rate

µ (0-24h)

Reduction (%)

µ (24-48h)

Reduction (%)

µ (48-72h)

Reduction (%)

control

0.03392

0.11509

0.05721

0.30

0.03961

-16.8

0.11270

2.1

0.05609

2.0

0.96

0.02854

15.9

0.11909

-3.5

0.05271

7.9

4.2

0.03624

-6.8

0.11309

1.7

0.05486

4.1

8.3

0.01518

55.3

0.12311

-7.0

0.05596

2.2

40

0.03814

-12.4

0.07792

32.3

0.05477

4.3

Determination of effect concentrations (based on TWA concentrations):

Parameter

 

TWA concentration

2-methylcyclohexyl acetate (mg/l)

95%-confidence interval (mg/l)

NOERC

4.2

 

72h-ERC10

15

5.7-38

72h-ERC50

>40

 

NOEYC

4.2

 

72h-EYC10

4.9

1.4-17

72h-EYC50

27

6.1-122

Validity criteria:

- In the controls, cell density increased by an average factor of > 16 within 2 days.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

- The mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35%. This high variation might have been due to the fact that the test vessels were completely filled with test medium which hinder a homogenous mixing of the low concentrations of the algae in the test medium at the start of the experiment. Prior to the other measurements algae were homogenized in the test medium by shaking this reduced the variation between the control vessels. It was considered that this deviation did not affect the integrity of the study result and the study was therefore considered valid.

Validity criteria fulfilled:
yes
Remarks:
(see above, "Any other information on results")
Conclusions:
The 72h-EC50 for growth rate reduction of 2-methylcyclohexyl acetate in Pseudokirchneriella subcapitata was > 40 mg/L TWA. The 72h-EC10 and 72h-NOEC were determined to be 15 mg/L and 4.2 mg/L respectively (based on growth rate, TWA concentrations).
Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with 2-methylcyclohexyl acetate. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, while three replicates per test group were exposed to nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/l in a static, air-tight closed system with an adjusted-M2 medium (extra sodium bicarbonate and HEPES buffer). The total test period was 72 hours and the initial algal cell density was 10E+04 cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. During the exposure period the concentrations did not remain constant (8.8-20% of initial) and therefore, effect parameters were reported as Time Weight Average (TWA) exposure concentrations. The range tested based on TWA concentrations was 0.30, 0.96, 4.2, 8,3 and 40 mg/l. 2-methylcyclohexyl acetate reduced growth rate and inhibited the yield significantly at concentrations of 8.3 mg/l and higher. The NOEC and the EC10 (based on growth rate) were 4.2 and 15 mg/L respectively. The 72 h-EC50 for growth rate reduction was determined to be > 40 mg/L. The 72h-EC50 for yield inhibition was 27 mg/l.

Description of key information

Key study: Test method OECD Guideline 201. GLP study. The 72h-EC50 of 2-methylcyclohexyl acetate in Pseudokirchneriella subcapitata for growth rate reduction was determined to be > 40 mg/L Time Weight Average (TWA). The NOEL and EC50 based where determined to be 4.2 and 15 mg/L respectively (basis for effect: growth inhibition, TWA).

Key value for chemical safety assessment

EC50 for freshwater algae:
40 mg/L
EC10 or NOEC for freshwater algae:
15 mg/L

Additional information

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with 2-methylcyclohexyl acetate. An initial algal cell denstity of 10E+04 were exposed for 72 hours to nominal concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/l in a static, air-tight closed system with an adjusted-M2 medium (extra sodium bicarbonate and HEPES buffer). During the exposure period the concentrations did not remain constant (8.8-20% of initial) and therefore, effect parameters were reported as Time Weight Average (TWA) exposure concentrations (0.30, 0.96, 4.2, 8,3 and 40 mg/l). 2-methylcyclohexyl acetate reduced growth rate and inhibited the yield significantly at concentrations of 8.3 mg/l and higher. The NOEC and the EC10 (based on growth rate) were 4.2 and 15 mg/L respectively. The 72 h-EC50 for growth rate reduction was determined to be > 40 mg/L. The 72h-EC50 for yield inhibition was 27 mg/l.