Reaction products of 2,4-bis(2-chlorophenyl)-5-(3,4-dimethoxy phenyl)-1H-imidazole and 2-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Test substance solubility and stability were not verified in the dilution water. Test substance solutions were not analyzed. Study endpoints are based on nominal test concentrations.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Test substance solubility and stability were not verified in the dilution water. Test substance solutions were not analyzed. Study endpoints are based on nominal test concentrations.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Purity: 100 wt% as a reaction product

Test solutions

Vehicle:

yes

Details on test solutions:

Test Solution Preparation

A primary stock solution with a nominal concentration of 100 mg/L test substance was prepared by dissolving approximately 50 mg of test substance and bringing this up to 500 mL in filter-sterilized AAP nutrient medium. Test solutions were prepared by adding the appropriate volume of the stock solution to filter-sterilized AAP nutrient medium to make nominal concentrations of 0.298, 0.954, 3.05, 9.77, and 31.3 mg/L test substance, and stirring each for approximately 5 minutes. Aliquots of the primary stock solution (which was sonicated for approximately 1 hour and stirred for 30 minutes) were used for the nominal 100 mg/L test concentration solution. Aliquots of the filter-sterilized AAP nutrient medium were used for the blank control solution. The blank control and test substance concentrations 0.298, 0.954, and 3.05 mg/L were clear and colorless with no visible precipitate at test start. Test substance concentration 9.77 mg/L was slightly cloudy with no visible precipitate; test substance concentration 31.3 mg/L was cloudy with no visible precipitate, and test substance concentration 100 mg/L was cloudy with precipitate present at test start.


AAP Nutrient Medium Preparation

To prepare one liter of AAP nutrient medium, 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution were added to approximately 800 mL of Milli-Q (deionized) water with mixing after each addition. The final volume of the medium was brought to 1 liter with additional Milli-Q water. The larger volume required for the definitive test was prepared based on these proportions. The nutrient medium pH was adjusted to 7.41 with 0.1 N hydrochloric acid and filter-sterilized through 0.22-µm cellulose acetate filters into sterile containers. The containers with the resulting filter-sterilized AAP nutrient medium were stored in the refrigerator in the dark at approximately 4ºC and acclimated to ambient temperature prior to use.

Test organisms

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata, a freshwater, unicellular, non-motile, green alga, used in this study was cultured and maintained at DuPont Haskell. The original culture source was the Department of Botany at the University of Texas at Austin.

The culture method for P. subcapitata was based on published literature. Prior to the study, cultures were maintained under photoperiod, shaking speed, and temperature conditions similar to those used in the study. Illumination was maintained at 5005 ± 805 lux. The organisms were cultured in sterilized 250-mL Erlenmeyer flasks containing approximately 50 mL of filter-sterilized synthetic algal-assay procedure (AAP) nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with sterilized foam stoppers to permit gas exchange. The P. subcapitata culture used to inoculate test vessels was aseptically transferred to fresh medium 4 days prior to use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21 to 24ºC controlled at ± 2ºC

pH:

7.13 to 8.03

Nominal and measured concentrations:

Nominal concentrations of 0.298, 0.954, 3.05, 9.77, 31.3, and 100 mg/L

Details on test conditions:

The test substance concentrations of 0.298, 0.954, 3.05, 9.77, 31.3, and 100 mg/L were chosen for the definitive test based on the results of a range finding test. Each test substance concentration was tested as 3 replicates. The blank control was 6 replicates. Flasks were inoculated with approximately 10,000 cells/mL by aseptically transferring 0.610 mL of algal inoculum from a pre-counted, logarithmically growing stock culture to each flask.

Test chambers were sterilized 250-mL Erlenmeyer flasks containing approximately 50 mL of test solution. Flasks were fitted with sterilized foam stoppers to permit gas exchange. Test flasks were randomly positioned on a shaker table with a shaking speed of approximately 100 rpm in an environmental chamber at 21 to 24ºC controlled at ± 2ºC. Air temperature, liquid temperature and pH were recorded. Flasks were illuminated continuously using cool-white fluorescent tubes.

Pseudokirchneriella subcapitata growth was determined by counting the number of cells in an approximate 0.2-mL sample from each flask at approximately 24, 48, and 72 hours after the definitive test initiation. The counts were conducted using a hemacytometer and a compound microscope. An aliquot of each sample was loaded into the 2 grid areas of the hemacytometer. All healthy cells located in 8 squares from each grid area (16 total squares) were counted and recorded in the study records. The total number of cells counted was multiplied by 10,000 to determine the number of cells per milliliter. Counts were made at approximately the same time as the day 0 inoculation.

Reference substance (positive control):

yes

Remarks:

Synthetic algal-assay-procedure (AAP) nutrient medium was used as the test diluent and blank (culture medium) control.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Basis for effect:

other: cell density and growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31.3 mg/L

Nominal / measured:

nominal

Basis for effect:

other: cell density and growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Basis for effect:

other: cell density and growth rate

Details on results:

All environmental parameters for the definitive test were within expected ranges. During the test, the shaking speed was 100 rpm, pH ranged from 7.13 to 8.03, mean lighting was 7242 lux with a range of 6820 to 7540 lux, and temperature in the environmental chamber was 23°C.

Cell density increased in the blank control by a factor of approximately 280 in 72 hours and the coefficient of variation of the average specific growth rate during the 72-hour exposure period in the blank control replicates was 4.0, and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the blank control replicates was 23.3, thereby satisfying the appropriate test acceptance criteria.

Inhibition of growth based on cell density of P. subcapitata exposed to nominal concentrations of 0.298, 0.954, 3.05, 9.77, 31.3, and 100 mg/L test substance for 72 hours was -16, -16, 22, 22, 5, and 29%, respectively. Inhibition of growth based on cell density yield was -16, -16, 22, 23, 5, and 30%, respectively. Inhibition of growth based on growth rate was -3, -3, 4, 4, 1, and 6%, respectively.

Reported statistics and error estimates:

Analyses are reported based on nominal test substance concentrations and were conducted using SAS Version 8.2. The data for cell density, cell density yield, and growth rate based on cell density were determined to be normally distributed (Shapiro-Wilk test) with equal variances (Levene’s test). Therefore, the Jonckheere-Terpstra trend test was used to determine the LOEC and NOEC values. No outliers, as determined by the Tukey outlier rule, were found in the data for cell density, cell density yield, and growth rate based on cell density. Massive ties, as determined by exact permutation data analysis methods, were present in the data for cell density, cell density yield, and growth rate based on cell density. However, exact permutation data analysis methods were not required. All statistical tests were calculated at a significance level of p = 0.05.

Any other information on results incl. tables

EC50, LOEC, and NOEC Values for Pseudokirchneriella subcapitata

Based on Exposure to Nominal Concentrations for 72 Hours

Parameter	EC50* (mg/L)	EC50 Model	LOEC (mg/L)	NOEC (mg/L)	LOEC and NOEC Method
Cell Density	>100	OECD model 2	100	31.3	Jonckheere-Terpstratrend test
Cell Density Yield	>100	OECD model 2	100	31.3	Jonckheere-Terpstratrend test
Growth Rate based on Cell Density	>100	OECD model 2	100	31.3	Jonckheere-Terpstratrend test

* 72-hour EbC50 for cell density; 72-hour EyC50 for cell density yield; 72-hour ErC50 for growth rate based on cell density, 95% confidence interval

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

EbC50 (72-hour) was >100 mg/L.
EyC50 (72-hour) was >100 mg/L.
ErC50 (72-hour) was >100 mg/L.
LOEC (72-hour) was 100 mg/L
NOEC (72-hour) was 31.3 mg/L

Executive summary:

The toxicity of the test substance to the green algae, Pseudokirchneriella subcapitata, was determined in a 72-hour, static toxicity test according to OECD Guideline 201 and OPPTS Guideline 850.5400.

 

The study was conducted with a blank control and 6 concentrations of test substance at a mean lighting intensity of 7242 lux (range of 6820 to 7540 lux), a mean air temperature of 23°C, a liquid temperature of 23°C, and a shaking speed of 100 rpm. Synthetic algal-assay-procedure (AAP) nutrient medium was used as the test diluent and blank (culture medium) control. Test solutions were not renewed. Three replicates were used per test concentration and 6 replicates were used for the control. Cell counts and growth rates were determined at 24-hour intervals over the 72-hour test.

 

Inhibition of growth expressed as cell count (density) ofPseudokirchneriella subcapitataexposed to nominal concentrations of 0.298, 0.954, 3.05, 9.77, 31.3, and 100 mg/L test substance for 72 hours was -16, -16, 22, 22, 5, and 29%, respectively. Inhibition of growth expressed as cell density yield was -16, -16, 22, 23, 5, and 30, respectively. Inhibition of growth expressed as the average specific growth rate was -3, -3, 4, 4, 1 and 6%, respectively. Negative values of inhibition indicate stimulation of growth. Cell density increased in the blank control by at least a factor of 16 in 72 hours, the coefficient of variation of average specific growth rates during the whole test period (0-72 hour) in the blank control replicates did not exceed 7%, and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the blank control replicates did not exceed 35%, thereby satisfying the appropriate test acceptance criteria.

 

EbC50 (72-hour) based on the 72-hour cell count (density) relative to the control was >100 mg/L.

EyC50 (72-hour) based on cell density yield was >100 mg/L.

ErC50 (72-hour) based on growth rate relative to the control was >100 mg/L.

LOEC (72-hour) based on the lowest concentration of test substance that had a significant effect on the measured parameter relative to the control was 100 mg/L (cell density, cell density yield and growth rate).

NOEC (72-hour) based on the highest concentration of test substance that had no significant effect on the measured parameter relative to the control was 31.3 mg/L (cell density, cell density yield and growth rate).

 

The most sensitive parameter was cell density with a 72-hour EbC50, EyC50, and ErC50 of >100 mg/L.