6-{4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenoxy}hexyl methacrylate

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:

Nov. 2016 - Feb. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: ROC-601
CAS Number: 439661-46-8
Appearance: White crystalline powder
Purity: 99.5 %
Storage: at room temperature, avoid exposure to light
Lot no: S23PSG0816
Expiration date: June 28, 2018

Analytical monitoring:

yes

Details on sampling:

ROC-601 content of the test solutions was measured at the start and at 24 hours intervals thereafter during the experiment. For determination of the test item concentration, at each analytical occasion six samples were taken from the test vessels and two samples from the control.
The analysis of the test item ROC-601 was performed by the analytical laboratory of
TOXI-COOP ZRT. according to the results of the analytical method validation (Study number: 888-100-1898) by HPLC method with UV detection.

Vehicle:

no

Details on test solutions:

For preparation of test solution a supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.1 g test item in 1000 mL test medium (OECD medium). This supersaturated solution was shaken for 20 hours and thereafter the non-dissolved test material was removed by filtration through a 0.22 µm membrane filter to obtain the saturated test solution (water accommodated fraction). The test solution was freshly prepared in the testing laboratory just before introduction of algae (start of the experiment). Untreated controls were run concurrently. The dilution water (OECD medium) without addition of test item was used as control solution.

Test organisms (species):

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

Details on test organisms:

Strain number: 61.81 SAG, (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
Pre-culturing: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for three days at this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations, as foreseen in the guidelines.

Hardness:

Standard OECD medium according to OECD 201 was used

Test temperature:

The temperature was in the range of 22.7 – 23.0 °C measured in the flask and between 21.4 and 23.3 °C measured within the climate chamber.

pH:

The pH was measured in the control and test item treated group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The pH of the control medium did not increase by more than 1.5 units during the test. The range of the pH was 8.10 – 9.18 during the experiment.

Salinity:

Standard OECD medium according to OECD 201 was used

Conductivity:

Standard OECD medium according to OECD 201 was used

Nominal and measured concentrations:

Nominal test concentration was 100 mg/L /water accomodated fraction). In the treated group mean of the measured concentrations was 2.03 µg/L at the start of the test. The mean measured test item concentration was below the LOQ (limit of quantification = 0.5 µg/L) at the 24h measurement and below the LOD (limit of detection = 0.2 µg/L) at the 48h and 72h measurements.

Details on test conditions:

The algal culture flasks were continuously illuminated with fluorescent lamps (with a spectral range of 400 - 700 nm). The light intensity was checked and recorded at the position occupied by algal culture flasks at the start of the test. The average measured light intensity was 8002 lux. The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.38 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: equivalent to > 100 mg/L nominal

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.38 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: equivalent to > 100 mg/L nominal

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.38 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: equivalent to > 100 mg/L nominal

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.38 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: equivalent to > 100 mg/L nominal

Details on results:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item ROC-601 had no toxic effect at aquatic saturation (i.e. water accommodated fraction, WAF) on the growth of green algae; the EC10, EC20, EC50 results and the LOEC (growth rate and yield) are higher than the solubility level of the test item in the test medium, measured as 0.38 µg/L which corresponds to a nominal concentration of 100 mg/L. Slight growth stimulation (negative inhibition) was observed in the treated group (-0.3 % inhibition for growth rates and -1.4 % for yield) after 72 hours of exposure compared to the control which was considered to be within the normal biological range based on the statistical evaluation. All validity criteria were met and therefore the study can be considered as valid.

Results with reference substance (positive control):

For the evaluation of the reliability of the applied test system and the experimental conditions Potassium dichromate is tested at least twice a year.
The date of the last study (Study Number: 392-201-1921) with the reference item Potassium dichromate was: 20 – 23 September 2016.
The 72h ErC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.77 mg/L)
The 72h EyC50: 0.46 mg/L, (95 % confidence limits: 0.42 – 0.49 mg/L)
Name and data of Reference Item
Name: Potassium dichromate (K2Cr2O7)
Batch No.: MKBN3524V
Description: Orange crystalline powder
Expiry Date: 04 March, 2018
Storage: Room temperature
Supplier: SIGMA-ALDRICH

Reported statistics and error estimates:

Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using 2 Sample t-Test (α = 0.05, TOXSTAT software). The results of the statistical evaluation showed that the 0 - 72 h average specific growth rate and yield at the treatment group (saturated test item concentration) was not statistically significantly different from the untreated control.

Preliminary Range-Finding Tests

In order to select appropriate test concentrations for use in the definitive test, two non-GLP preliminary range-finding tests were conducted to determine the approximate toxicity of the test item.

Preparation of test solutions was performed using the WAF method (according toOECD Series on Testing and Assessment No. 23) equivalent as in the main study(nominal 100 mg/L). The stock solution was then filtrated through a membrane filter (0.22 µm) to separate the possible non-dissolved test material. The further test solutions were prepared from this stock solution. Untreated control ran parallel in the test.

Algal cells were exposed to each concentration of the test item plus control, for 72 hours. The first pre-test was performed with two replicates in treatment groups and three replicates in the control, while in the second pre-test three control replicates and six test item treated replicates were included.

The concentration levels (loading rate) used and results (72-h) of thepre-experimentare summarised in the following tables:

Results of the First PreliminaryTest

Test Concentration (mg/L)	Untreated control	1	10	100
Average of cell number at 72 hours (x 10^4cells/mL)	49.44	47.50	49.33	41.17
Growth Rates(µ) [0-72h]	0.0542	0.0536	0.0541	0.0516
% Inhibition ofµ [0-72h]	-	1.0	0.1	4.7
Yield(Y) [0-72h]	48.44	46.50	48.33	40.17
% Inhibition ofY [0-72h]	-	4.0	0.2	17.1

 

Results of the Second PreliminaryTest

Test Concentration (mg/L)	Untreated control	100
Average of cell number at 72 hours (x 10^4cells/mL)	129.22	127.72
Growth Rates(µ) [0-72h]	0.0673	0.0671
% Inhibition ofµ [0-72h]	-	0.3
Yield(Y) [0-72h]	128.22	126.72
% Inhibition ofY [0-72h]	-	1.2

Based on the results of thenon-GLP Preliminary Range-Finding Tests (see above), the main test included a nominal concentration of 100 mg/L (limit test) and a concurrent control group.

Performance of the Main Test

The test was started (0 hours) by inoculation of 0.1 mL algal biomass (10E7 algalcells per mL) to 100 mL test item solution. The initial cell density was approximately 10E4 cells/mL in each test flask. The test was performed with six replicates per test concentration and control. Furthermore, three replicate test vessels and one control flask were included (containing alga inoculum) for investigation of analytical measurements parallel with the study. Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in 250 mL Erlenmeyer flasks. The test flasks were covered with air-permeable stoppers. The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. The cell morphology was examined in parallel. The environmental conditions were recorded as described. For determination of the test item concentration, at each analytical occasion six samples were taken from the test vessels and two samples from the control.

Assay Acceptance Criteria

The cell concentration in the untreated control cultures should have increased by a factor of at least 16 within three days (72 hours), corresponding to a specific growth rate of 0.92/day. The cell density in the control cultures increased by a factor of 108.17 within 72 hours. This corresponds to a specific growth rate of 1.56 day/d.

The mean coefficient of variation for section-by-section specific growth rates (days 0 - 1, 1 - 2 and 2 - 3, for 72-h tests) in the control cultures must not exceed 35%.

The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0 - 1, 1 - 2 and 2 - 3, for 72 h-tests) in the control cultures did not exceed 35 %

- CV for section-by-section growth rate day 0 - 1: 5.64 %

- CV for section-by-section growth rate day 1 - 2: 7.26 %

- CV for section-by-section growth rate day 2 - 3: 6.70%

The mean coefficient of variation for section-by-section specific growth rates was 6.53 %.

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7%. The coefficient of variation of average specific growth rates during the whole test period (day 0 - 3) in the control cultures was 2.01 %.

All validity criteria were met and thus the test was considered valid.

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the results showed that the test item ROC-601 had no toxic effect at aquatic saturation (i.e. water accommodated fraction, WAF) on the growth of green algae; the EC10, EC50 results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to a nominal concentration of 100 mg/L (0.38 µg/L, measured). Thus, the EC50 is >0.38 µg/L and the NOEC is 0.38 µg/L.

Executive summary:

The effect of ROC-601 test item was assessed on algal growth using the unicellular green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), over an exposure period of 72 hours.

A limit test was performed in which, algal cells were exposed to aqueous test media containing the test item for 72 hours at the limit of its solubility in the test medium (i.e.water accommodated fraction, WAF) plus a control in order to demonstrate that the test item has no influence on the growth of algae up to at least the saturation concentration.

The quantification of the test itemROC-601was performed by a validated analytical method. Samples were taken from the test concentration and the control at the start and during 24 hour intervals thereafter during the experimentand analysed byHPLC method with UV detection. The determined exposure concentration of the test item was 0.38 µg/L which was calculated as the geometric mean of the measured daily concentrations.

Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates per test concentration and control. Furthermore, three replicate test vessels and one control flask were included (containing alga inoculum) for investigation of analytical measurements parallel with the study. The alga cell concentration was approximately 10E4 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by microscope in each testing flask during the test, in 24-hour intervals.

The results of the statistical evaluation showed that the 0 - 72 h average specific growth rate and yield in the treatment group (saturated test item concentration) was not statistically significantly different from the untreated concurrent control.

All validity criteria were met and therefore the study can be considered as valid.

 

Summary of the Biological Endpoints

Endpoints (0-72 h)	Growth rate (µ)	Yield (y)
EC10	> 0.38 µg/L (equivalent to > 100 mg/L nominal)	> 0.38 µg/L (equivalent to > 100 mg/L nominal)
EC20	> 0.38 µg/L (equivalent to > 100 mg/L nominal)	> 0.38 µg/L (equivalent to > 100 mg/L nominal)
EC50	> 0.38 µg/L (equivalent to > 100 mg/L nominal)	> 0.38 µg/L (equivalent to > 100 mg/L nominal)
NOEC	0.38 µg/L (equivalent to > 100 mg/L nominal)	0.38 µg/L (equivalent to > 100 mg/L nominal)
LOEC	> 0.38 µg/L (equivalent to > 100 mg/L nominal)	> 0.38 µg/L (equivalent to > 100 mg/L nominal)

 

In conclusion in this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item ROC-601 had no toxic effect at aquatic saturation (i.e.water accommodated fraction, WAF) on the growth of green algae; theEC_10,EC₅₀ results and the LOEC are higher than the solubility level of the test item in the test medium, which corresponds to a nominal concentration of 100 mg/L.

Description of key information

In a 72-h OECD 201 algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item ROC-601 had no toxic effect at aquatic saturation (i.e.water accommodated fraction, WAF) on the growth of green algae; the EC₁₀, EC₅₀ results and the LOEC are higher than the solubility level of the test item in the test medium (0.38 µg/L), corresponding to a nominal concentration of 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.38 µg/L

EC10 or NOEC for freshwater algae:

0.38 µg/L