reaction mass of cis 4-(3-methylbutyl)cyclohexanol and trans 4-(3-methylbutyl)cyclohexanol

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:

2008-10-07 to 2008-11-06

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2007-12-07

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Symrose
- Substance type: technical product
- Physical state: colourless to pale yellow liquid
- Storage condition of test material: ambient temp., dark and dry
No further details are given.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No further test substance was used.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The determination of the active substance in water samples was performed with a suitable analytical method (GC-MS).
- Concentrations: 0.102, 0.256, 0.64, 1.6, 4, 10 and 25 mg/L
- Sampling method: The concentration course of Symrose was verified in test medium by analysing the contents in the samples over the whole test period in intervals of 24 hours. Samples were taken after initiation of the test and thereafter in 1 day intervals until the end of the test after 72 hours at the concentration levels of 1.6, 4, 10 and 25 mg/L and control.

Due to the fact that the EC0 was 4 mg/L and the EC100 10 mg/L it was decided not determining the actual analytical concentration of the lower test concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

no

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: SAG 86.81
- Source (laboratory, culture collection): cultures of the "Pflanzenphysiologisches Institute der Universität Göttingen"
- Method of cultivation: The algae are grown in semi-continuously in the laboratory in aerated liquid cultures under permanent illumination. Old medium was periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Culture conditions were as followed: 22-25°C, 1000mL screw cap bottles as culture flasks, CO2 supply ba aeration and illumination with OSRAM light tubes (approx 7000 lux).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Hardness:

no data

Test temperature:

23-24°C

pH:

t = 0: 7.42 - 7.72
t = 72: 8.24 - 8.95

Dissolved oxygen:

no data

Salinity:

NH4Cl 15 mg/L
MgCl2 x 6 H2O: 12 mg/L
CaCl2 x 2 H2O: 18 mg/L
MgSO4 x 7 H2O: 15 mg/L
KH2PO4: 1.6 mg/L
FeCl3 x 6 H2O: 0.08 mg/L
Na2EDTA x 2H2O: 0.1 mg/L
H3BO3: 0.185 mg/L
MnCl2 x 4 H2O: 0.415 mg/L
ZnCl2: 3 µg/L
CoCl2 x 6H2O: 1.5 µg/L
CuCl2 x 2H2O: 0.01 µg/L
Na2MoO4 x 2H2O: 7 µg/L
NaHCO3: 50 mg/L

Nominal and measured concentrations:

nominal concentrations: 1.6, 4, 10 and 25 mg/L (spaced by the factor 2.5)
measured concentrations: 1.2, 2.2, 9.1 and 26.1 mg/L (based on geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with aluminium caps and two baffles
- Material, size, headspace, fill volume: glass, 500 mL flask with a final volume of approximately 167 mL in each test vessel
- Initial cells density: 0.5 x 10^4
- Control end cells density: 120.52 x 10^4
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Light intensity and quality: 400W, approx. 7000 lux (continuous illumination)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: fluorescence detection after 24, 48 and 72 hours of growth

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 0, 0.01, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: inhibitory effects were observed from 1 to 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.85 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.96 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 7.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 10

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 3.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

for individual results see attached tables

Results with reference substance (positive control):

Results after 3 days test duration with K2Cr2O7 (Batch No.: K29220264 141).
- EC50 cell number: 0.49 mg/L
- EC50 growth rate: 1.56 mg/L
- EC50 biomass integral: 0.41 mg/L
- EC50 yield: 0.41 mg/L
- LOEC: 0.256 mg/L
- NOEC: 0.1024 mg/L

Reported statistics and error estimates:

To estimate the LOEC, and hence the NOEC, ANOVA was used to calculate the mean average specific growth rate for each test concentration. The resulting mean for each test concentration was compared with the control mean (all controls pooled, including control) using an the comparison method of Dunnett’s test (DUNNETT, 1964). Before pooling data for the control, they were statistically tested to see that they are not significantly different, using two-tailed t-test.
A test for normality of the data was done by calculating the Shapiro-Wilk’s statistic.

Any other information on results incl. tables

The validation of the analytical method was performed in accordance with SANCO/3029/99 rev. 4 (11/07/00):

Linearity: The analytical system gave linear response in the calibration range of 0.1 to 20 μg/mL. The correlation coefficient (r) of the calibration curves was 0.9999.

LOQ: The LOQ of the analytical method was fixed at nominal 0.8 mg/L of the test item and checked by means of accuracy

The mean recovery rate (fortification level 0.8 and 100 mg/L n = 5) was between 97 and 99% with a RSD between 4.8 and 6.2%. The response of blank values of test medium control samples was lower than 30 % of LOQ.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see executive summary

Conclusions:

Despite the loss of test material during the test, there is no drastic change in the toxicological assessment. Symrose was found to be toxic to algae after 72 hours (1 mg/L < EC50 < 10mg/L).

Symrose was found to be toxic to Daphnia magna after 48 hours. The EC50 (48 hours) was 7.86 (7.37 – 8.38) mg/L. The NOEC after 48 hours was 1.40 mg/L. The LOEC after 48 hours was 3.06 mg/L.

Executive summary:

The test fulfils the validity criteria in accordance with OECD Guideline for Testing of Chemicals No. 201, since

(i) cell numbers, measured in the controls between 0 h and 72 h, were found to increase by a factor of 241 which exceeds the threshold of 16, this corresponds to a growth rate of 1.83 · d^-1

(ii) the coefficient of variation of average growth in replicate control cultures did not exceed 7 % for the whole test period

(iii) The mean coefficient of variation for the section-by-section specific growth rates (days 1-2 and 2-3) in the control cultures did not exceed 35% (e.g. 24 % and 7 %), but the mean coefficient of variation for the section-by-section specific growth rate for days 1-2 exceeded 35 % (105 %).

However, the section-specific growth rate of replicate number 1 (out of 6 replicates) is statistically an outlier (smaller than 2 standard deviations below the mean) in this section (Days 0-1) ONLY, but not in the other sections.The coefficient of variation for the section-by-section specific growth rates (days 0-1) is calculated to be 13% without replicate 1 (n=5). Considering that fact and that the growth rates for other sections comply with the guideline criteria it can be concluded that the vailidity criteria of the study are fulfilled (see attachment "raw data").