Decahydro-2-naphthyl acetate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 December 2016 to 23 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

In the first study, the chosen range of concentrations did not cover all EC and NOEC/LOEC levels. Therefore, a second study was carried out to test additionally a lower range of concentrations.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations and the control were analytically verified at the start and end (72 hours) of exposure.
- Sampling method: Samples at the start of exposure were taken from separate replicates prepared without algae. Samples after 72 hours were prepared with algae and incubated under the test conditions.
- Sample storage conditions before analysis: All samples were stored were stored at 6 ± 2 °C until sample preparation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 100 mg/L test item was prepared once with demineralised water 24 ± 1 hour prior to the start of the exposure. The test item was placed on to the surface of the demineralised water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 24 ± 1 hour. After a separation phase of at least 1 hour (for the 2nd study only 5 minutes) at room temperature the saturated solution was taken from the homogeneous phase and was used for testing. Then the components of the dilution water were added to the saturated solution. The saturated solution and dilution levels of the saturated solution were used as the test concentrations.
- Controls: Blank control
- Evidence of undissolved material: The saturated solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: HINDÄK, SAG 61.81
- Age of inoculum: 4 days old
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar.

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions: Algae were cultured in nutrient medium Z according to Lüttge et al. (1994) under continuous light (2590 to 5180 lux).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

1st study: 22.0 to 22.5°C
2nd study: 22.0 to 23.5°C

pH:

1st study test start: 7.98 to 8.06
1st study test end: 8.09 to 9.49
2nd study test start: 8.06 to 8.12
2nd study test end: 8.99 to 9.32

Nominal and measured concentrations:

1st study nominal concentrations: 6.25, 12.5, 25.0, 50.0, 100 mg/L
1st study geometric mean measured concentrations: 1.73, 3.12, 6.53, 14.7 and 32.5 mg/L
2nd study nominal concentrations: 1.56, 3.13, 6.25 mg/L
2nd study geometric mean measured concentrations: 0.496, 1.17, 1.91 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks (volume 59 mL) with aluminum tops with PTFE seals
- Type: Closed
- Material, size, headspace, fill volume: 59 mL
- Initial cells density: 6305 cells/mL in 1st study, 6839 cells/mL in 2nd study
- Control end cells density: 688858 cells/mL in 1st study, 536756 cells/mL in 2nd study.
- No. of vessels per concentration (replicates): 3, separate replicates for each measuring time were prepared.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD TG 201 medium

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Nutrient medium Z according to Lüttge et al. (1994) as culture medium.
- Intervals of water quality measurement: The pH-value at the start of exposure was measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-value was measured from pooled samples of each test item concentration and the control after measurement of the cell density. The room temperature was measured continuously. Light intensity was measured prior to the start of the exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous light
- Light intensity and quality: 4440 to 8880 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Fluorometer; the cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.
- Other: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes
- Test concentrations: 1.00, 10.0 and 100% saturated solution (measured concentrations after 72 hours 0.256, 2.41 and 23.5 mg/L)
- Results used to determine the conditions for the definitive study: Growth rate inhibitions were -3, 2 and 100 % for 1.00, 10.0 and 100% saturated solutions, respectively, and yield inhibitions were -12, 10 and 100%, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 6.05 - 7.01 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 2.62 - 2.96 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.63 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 2.14 - 3.21 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.814 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: <0.496 - 1.24 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.496 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.559 (with headspace) and 0.811 (without headspace) mg/L for growth rate, 0.320 (with headspace) and 0.387 (without headspace) for yield.
- Other: Effect levels were determined with and without headspace.

Reported statistics and error estimates:

EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The data of both parts were analysed together. The NOEC and LOEC for the 1st study were determined using multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm for growth rate, Williams Multiple t-test for yield and Monotonicity of Concentration/Response for yield by trend analysis by contrasts, all at a significance level of 0.05. For the 2nd study, the NOEC and LOEC were calculated using One Way Analysis of Variance (ANOVA) and Dunnett’s test (α = 0.05). Data were tested for normality and homogeneity before applying the statistical tests.

Table 1. Cell densities

Geometric mean measured concentrations (mg/L)	Mean cell density (cells/mL)
	0 hours	24 hours	48 hours	72 hours
1st study
Control	6305	35390	21355	688858
1.73	6305	28963	163957	542514
3.12	6305	16464	51416	267887
6.53	6305	12951	28323	81009
14.7	6305	6702	6706	4400
32.5	6305	< LOQ	< LOQ	< LOQ
2nd study
Control	6839	24707	136116	536756
0.496	6839	22310	126417	487273
1.17	6839	21597	131600	463484
1.91	6839	20459	115499	419818

LOQ = 2763 cells/mL

Table 2. Inhibition of growth rate and yield results

Geometric mean measured concentrations (mg/L)	Growth rate (/d)	Inhibition of growth rate (%)	Yield (cells/mL)	Inhibition of yield (%)
1st study
Control	1.56	-	682553	-
1.73	1.48*	5	536209*	21
3.12	1.25*	20	261582*	62
6.53	0.84*	46	74704*	89
14.7	-0.14*	100	-1905*	100
32.5	n/a*	100	n/a*	100
2nd study
Control	1.45	-	529917	-
0.496	1.42	2	480434	9
1.17	1.41*	3	456645*	14
1.91	1.37*	6	412979*	22

Statistically significant differences of growth rates and yield compared to control values are marked *.

n/a = data not determinable

Validity criteria fulfilled:

yes

Remarks:

Exponential cell growth observed in the controls, the mean coefficients of variation for section-by-section specific growth rate were ≤ 35% and the coefficients of variation of average specific growth rates were ≤ 7% in the controls.

Conclusions:

The EC50-values for inhibition of growth rate and yield after 72 hours were 6.51 mg/L and 2.78 mg/L, respectively, and the EC10-values were 2.63 mg/L and 0.814 mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.496 mg/L.

Executive summary:

The toxicity of the test item to the green alga (Pseudokirchneriella subcapitata) was determined in a 72 -hour static test according to OECD Guideline 201 and EU Method C.3. In the first study, the chosen range of concentrations did not cover all EC and NOEC/LOEC levels. Therefore, a second study was carried out to test additionally a lower range of concentrations. Nominal concentrations of the test item in the first study were 6.25, 12.5, 25.0, 50.0, 100 mg/L (corresponding to geometric mean measured concentrations of 1.73, 3.12, 6.53, 14.7 and 32.5 mg/L, respectively) and nominal concentrations in the second study were 1.56, 3.13, 6.25 mg/L (corresponding to geometric mean measured concentrations of 0.496, 1.17, 1.91 mg/L, respectively). A saturated solution of the test item was prepared using a slow stirring procedure and diluted to make up the test solutions. Based on geometric mean measured concentrations, the EC50-values for inhibition of growth rate and yield after 72 hours were 6.51 mg/L and 2.78 mg/L, respectively, and the EC10-values were 2.63 mg/L and 0.814 mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.496 mg/L. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was performed according to guideline methods.

Description of key information

Based on geometric mean measured concentrations, the EC50-values for inhibition of growth rate and yield after 72 hours were 6.51 mg/L and 2.78 mg/L, respectively, and the EC10-values were 2.63 mg/L and 0.814 mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.496 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

6.51 mg/L

EC10 or NOEC for freshwater algae:

2.63 mg/L

Additional information

The toxicity of the test item to green alga (Pseudokirchneriella subcapitata) was determined in a 72 -hour static test according to OECD Guideline 201 and EU Method C.3 (2017). In the first study, the chosen range of concentrations did not cover all EC and NOEC/LOEC levels. Therefore, a second study was carried out to test additionally a lower range of concentrations. Nominal concentrations of the test item in the first study were 6.25, 12.5, 25.0, 50.0, 100 mg/L (corresponding to geometric mean measured concentrations of 1.73, 3.12, 6.53, 14.7 and 32.5 mg/L, respectively) and nominal concentrations in the second study were 1.56, 3.13, 6.25 mg/L (corresponding to geometric mean measured concentrations of 0.496, 1.17, 1.91 mg/L, respectively). A saturated solution of the test item was prepared using a slow stirring procedure and diluted to make up the test solutions. Based on geometric mean measured concentrations, the EC50-values for inhibition of growth rate and yield after 72 hours were 6.51 mg/L and 2.78 mg/L, respectively, and the EC10-values were 2.63 mg/L and 0.814 mg/L, respectively. The NOEC-values for inhibition of both growth rate and yield after 72 hours were 0.496 mg/L. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was performed according to guideline methods.