cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

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:

2015-09-21 to 2015-10-29

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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

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: I14KB4717
- Expiration date of the lot/batch: 206-11-25 (retest date)
- Purity test date: 2014-12-13 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test and after 72 hours from all test concentrations and from the control. A volume of 2.0 mL was taken. The filter used for preparation of the SS was kept for possible analysis of the residue. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples of 2.0 mL were taken from all test solutions for possible analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Combined limit/range-finding test:
Based on the data provided by the Sponsor, the solubility of the test substance in water was 39.3 g/L. Because in the parallel studies with Daphnia magna and carp (Projects 508946 and 508948) after magnetic stirring in ISO medium flocculation was observed, it was decided to prepare the highest concentration in Milli-RO water and subsequently add salts. A loading rate of 100 mg/L was prepared in Milli-RO water, applying a 45-minute period of magnetic stirring after which the solution was clear and colourless. Subsequently salts were added to reach the concentrations described in report paragraph 6.3 (M2 medium) and used as the highest concentration. 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 at the start of the test except for the highest concentration that was hazy and contained undissolved particles.

Final test:
Based on the observations made in the combined limit/range-finding test and in agreement with the sponsor, the solutions for the final test were prepared differently. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L applying a one-hour period of magnetic
stirring. The obtained mixture was allowed to settle for 57 minutes (at room temperature) and subsequently was filtered through a preconditioned membrane filter (RC55, Whatman). The obtained SS was used as the stock solution. The lower test concentrations were prepared by subsequent
dilutions of the stock solution in test medium. The final test solutions were all clear and colourless at the start of the test.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.

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: Algal stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.


ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

19-20°C

pH:

8.0-8.2

Dissolved oxygen:

not reported

Nominal and measured concentrations:

Range-finder:
nominal test concentrations: control, 0.1, 1, 10, 100 mg/L (only 0.1, 100 mg/L measured)
measured concentrations: t= 0 h: 0.10, 91.2 mg/L
measured concentrations: t= 24 h: 0.09, 38.3 mg/L
measured concentrations: t= 48 h: 0.09, 66.7 mg/L

Final test:
nominal test concentrations final test: 0, 0.032, 0.10, 0.32, 1.0, 3.2, 10 and 32% of a Saturated Solution prepared at 100 mg/L
measured test concentration final test t= 0 h: n.d., 0.021, 0.059, 0.184, 0.588, 0.595, 1.86, 5.65, 15.6 mg/L
measured test concentration final test t= 24h: n.d., 0.025, 0.061, 0.185, 0.593, 0.602, 1.91, 5.78, 17.7 mg/L
measured test concentration final test t= 72h: 0.018, 0.024, 0.061, 0.181, 0.578, 0.552, 1.89, 5.89, 16.4 mg/L

These measured concentrations remained stable during the exposure period (98-114% of initially measured at the end of the test). The effect parameters were
expressed in terms of initially measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 124.1 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, 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.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study:
- Test concentrations: 1.00, 10 and 100 % of the SS
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

5.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL 95% 5.2-6.7 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.059 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- EC50 (yield) = 1.1 mg T001202 /L
- EC10 (growth rate) = 0.26 mg T001202 / L (95% C.L. 0.21-0.30 mg/L).
- EC10 (yield) = 0.02 mg T001202 / L (95% C.L. 0.01-0.03 mg/L).
- NOEC (yield) = 0.021 mg T001202 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L

Reported statistics and error estimates:

Calculation of ECx values was based on Weibull (Growth rate inhibition) or probit (yield inhibition) analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding measured concentrations of the test item.
The calculations were performed with ToxRat Professional v. 3.2.0. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed wit the test substance T001202 according to the OECD guideline 201 (GLP conditions).
Under the conditions of the present study with Pseudokirchneriella subcapitata, T001202 reduced growth rate of this fresh water algae species significantly at a measured concentration of 1.9 mg/L and higher. The EC50 for growth rate inhibition (72h-ErC50) was 5.9 mg/L. The 95% confidence limit ranged from 5.2 to 6.7 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tobor-Kaplon (2016), investigating the acute toxicity of T001202 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h NOEC and 72-h EC50 for growth rate were determined to be 0.059 mg/L and 5.9 mg/L respectively. The substance can be considered as harmful to algae

Key value for chemical safety assessment

EC50 for freshwater algae:

5.9 mg/L

EC10 or NOEC for freshwater algae:

0.059 mg/L