Diallyl hexahydrophthalate

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:

13 December 2016 - 21 August 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)

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source from the sponsor, batch/lot number: 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date: 99.2%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%), protected from light
- Stability under test conditions: n/a
- Solubility and stability of the test substance in the solvent/vehicle: At least 3 days at 5 +/- 3°C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:A stock solution with a nominal concentration of 100.0 mg/L was prepared with the test item and algal growth medium (OECD Medium) using ultrasonic bath (~ 32 minutes).
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: Stock solution with nominal concentration of 100.0 mg/L
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

OTHER SPECIFICS: n/a

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Measured geometric mean test item concentrations were: 1.06, 3.34, 10.21, 30.85 and 104.0 mg/L.
- Sampling method: Samples were taken from the test solutions and one sample from the control solution. Samples were properly diluted and analysed by an HPLC-UV method.
- Sample storage conditions before analysis: not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution with a nominal concentration of 100.0 mg/L was prepared with the test item and algal growth medium (OECD Medium) using ultrasonic bath (~ 32 minutes). The test solutions were prepared by the appropriate diluting of this stock solution with dionised water and distributed into test vessels prior to introduction of algae
- Eluate: not specified
- Differential loading: not specified
- Controls: Untreated control - Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations. Reference control - potassium dichromate used to demonstrate satisfactory test conditions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not specified
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not specified

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Age of inoculum (at test initiation): not specified, but stock cultures are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
- Method of cultivation: The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

ACCLIMATION
- Acclimation period: not specified
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: not specified, but deformed or abnormal cells are discarded.

Test type:

not specified

Water media type:

freshwater

Remarks:

Deionised water used

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not specified

Test temperature:

The temperature was in the range of 22.5 – 23.1 °C measured in the flask, and between 21.9 and 23.6 °C measured within the climate chamber.

pH:

The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.57 – 8.81 during the experiment.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100 mg/L.
The corresponding measured geometric mean test item concentrations were: 1.06, 3.34, 10.21, 30.85 and 104.0 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): not specified
- Initial cells density: 10^4 algal cells per mL test medium
- Control end cells density: not specified
- No. of organisms per vessel: 10^4 algal cells per mL test medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
- Total organic carbon: not specified
- Particulate matter: not specified
- Metals: not specified
- Pesticides: not specified
- Chlorine: not specified
- Alkalinity: not specified
- Ca/mg ratio: not specified
- Conductivity: not specified
- Culture medium different from test medium:
- Intervals of water quality measurement: Begiining and end of the test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: not specified
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8373 lux (equivalent to
~113 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400- 700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Chlorophyll measurement: not specified
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not specified
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 0.1, 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: Yes (because significant inhibition was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 3.2 and one control were tested in the main experiment).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Growth rate (r)

Effect conc.:

ca. 23.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Growth rate (r)

Effect conc.:

ca. 3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Growth rate (r)

Effect conc.:

ca. 9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Yield (y)

Effect conc.:

ca. 9.61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Yield (y)

Effect conc.:

ca. 3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Yield (y)

Effect conc.:

ca. 9.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Biomass (b)

Effect conc.:

ca. 9.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Biomass (b)

Effect conc.:

ca. 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Biomass (b)

Effect conc.:

ca. 3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): not specified
- Unusual cell shape: not specified
- Colour differences: not specified
- Flocculation: not specified
- Adherence to test vessels: not specified
- Aggregation of algal cells:not specified
- Other:
- Any stimulation of growth found in any treatment: not specified
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
- Effect concentrations exceeding solubility of substance in test medium: not specified

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: EC50 growth = 0.88 mg/L. EC50 yield = 0.54 mg/L. EC50 biomass = 0.64 mg/L
- Other:

Reported statistics and error estimates:

Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

Growth rate - the 72 h EC50 value was determined [by Probit analysis (TOXSTAT software)] as 23.94 mg/L (95 % confidence limits: 20.44 – 28.04 mg/L).

Yield - The 72 h EC50 value was determined [by Probit analysis (TOXSTAT software)] as 9.61 mg/L (95 % confidence limits: 8.31 – 11.10 mg/L).

Biomass - The 72 h EC50 value was determined [by Probit analysis (TOXSTAT software)] as 9.85 mg/L (95 % confidence limits: 8.33 – 11.65 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates and yield were significantly different from that of the control group in the examined concentration range of 9.8 – 100.0 mg/L (nominal), therefore the NOEC was determined as 3.1 mg/L (nominal) and LOEC was determined as 9.8 mg/L (nominal). The 0-72 h areas were significantly different from that of the control group in the examined concentration range of 3.1 – 100.0 mg/L (nominal). The NOEC was determined as 1.0 mg/L (nominal); the LOEC was determined as 3.1 mg/L (nominal).

Executive summary:

The purpose of the study was to determine the effect of the test item MDAC on the growth of the unicellular green algal species Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) over an exposure period of 72 hours. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions, with nominal test item concentrations of 1.0, 3.1, 9.8, 31.3 and 100 mg/L, and the corresponding measured geometric mean test item concentrations were: 1.06, 3.34, 10.21, 30.85 and 104.0 mg/L. The test design included three replicates at each test concentration and six replicates for the untreated controls.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates and yield were significantly different from that of the control group in the examined concentration range of 9.8 – 100.0 mg/L (nominal), therefore the NOEC was determined as 3.1 mg/L (nominal) and LOEC was determined as 9.8 mg/L (nominal). The 0-72 h areas were significantly different from that of the control group in the examined concentration range of 3.1 – 100.0 mg/L (nominal). The NOEC was determined as 1.0 mg/L (nominal); the LOEC was determined as 3.1 mg/L (nominal).

Description of key information

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. Following the results from the preliminary range-finding test, the nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100 mg/L. Test concentrations were analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test item concentrations were: 1.06, 3.34, 10.21, 30.85 and 104.0 mg/L.

A stock solution with a nominal concentration of 100.0 mg/L was prepared with the test item and algal growth medium (OECD Medium) using an ultrasonic bath (~ 32 minutes). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae. For the untreated control, algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations. Potassium dichromate was used as a reference control, being tested on 07 -10 March 2017 where all values were within the range of laboratory ring test data.

The test was started (0 hours) by inoculation of a biomass of approximately 10⁴ algal cells per mL test medium. The test was performed with three replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers. The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Analytical measurement was performed at the applied test concentration levels and from the control at the beginning and at the end of the experiment. The samples were analysed by HPLC-UV method.

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

The 72 h E_rC₅₀ value was determined [by Probit analysis (TOXSTAT software)] as 23.94 mg/L (95 % confidence limits: 20.44 – 28.04 mg/L). The 72 h E_bC₅₀ value was determined [by Probit analysis (TOXSTAT software)] as 9.85 mg/L (95 % confidence limits: 8.33 – 11.65 mg/L). The 72 h E_yC₅₀ value was determined [by Probit analysis (TOXSTAT software)] as 9.61 mg/L (95 % confidence limits: 8.31 – 11.10 mg/L).

CONCLUSION:

The effect of MDAC test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata(formerly known as Selenastrum capricornutum), over an exposure period of 72 hours. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates and yield were significantly different from that of the control group in the examined concentration range of 9.8 – 100.0 mg/L (nominal), therefore the NOEC was determined as 3.1 mg/L (nominal) and LOEC was determined as 9.8 mg/L (nominal). The 0-72 h areas were significantly different from that of the control group in the examined concentration range of 3.1 – 100.0 mg/L (nominal). The NOEC was determined as 1.0 mg/L (nominal); the LOEC was determined as 3.1 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

23.94 mg/L

EC10 or NOEC for freshwater algae:

3.1 mg/L