Prometryn

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Oct 1992 to 4 Nov 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA - FTFRA Pesticide Assessment Guidelines Subdivision J, Hazard Evaluation: Non-Target Plants Guideline 123-2

Version / remarks:

Growth and Reproduction of Aquatic Plants, Tier 2

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were analyzed for the actual concentration of the test substance present in the test solutions on day 0 and day 5. Samples for the day 0 analyses were portions of the test treatments used to begin the test. At test termination, the contents of the replicate flasks were combined, the pH was recorded and the algae were removed by centrifugation at 3700 rpm for four minutes. The supernatants were then submitted for analyses.

Vehicle:

yes

Remarks:

N,N-dimethylformamide (DMF)

Details on test solutions:

A primary stock solution of 1.02 mg a.i./mL in the solvent N,N-dimethylformamide (DMF) was prepared by weighing 10.4 mg of the test substance to the nearest 0.1 mg, and diluting to 10.0 mL in a volumetric flask with DMF. The stock solution was mixed by inverting several times, resulting in a clear liquid. Other stock solutions (0.0005, 0.001, 0.002, 0.004, 0.008 and 0.016 mg/mL in DMF) were prepared by serial dilution.

Test concentrations were prepared by adding 0.25 mL of the appropriate stock solution to AAP/Si medium in 500 mL volumetric flasks to yield nominal test concentrations of 0.250, 0.500, 1.00, 2.00, 4.00, and 8.00 µg/L, respectively. The no-treatment control contained medium only. A solvent control was prepared to contain the same amount of DMF that was added to each test treatment, i.e. 0.5 mL/L. After thorough mixing, 50 mL of the no-treatment control, solvent control and each test treatment were added to each of four replicate test vessels.

Test organisms (species):

Navicula pelliculosa

Details on test organisms:

TEST ORGANISM
- Source: laboratory stock

MAINTAINED CONDITION
- Medium: Stock cultures were maintained in synthetic algal assay procedure nutrient medium with silicon
- Light intensity: 4306 lux (400 foot-candles)
- Temperature: 24 ± 2°C
- Shaking speed: 100 oscillations per minute
- Transfers were made regularly into fresh medium to provide cultures in the logarithmic phase of growth for toxicity test inoculations.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

5 d

Test temperature:

24 ± 2°C

pH:

- Day 0: 7.29 - 7.37
- Day 5: 7.17 - 7.63

Nominal and measured concentrations:

Nominal concentration: Negative control, solvent control, 0.250, 0.500, 1.00, 2.00, 4.00 and 8.00 µg/L
Measured concentration: N.D., N.D, 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µg/L, respectively. See Table 1 in "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: Closed with foam stoppers to permit gas exchange
- Initial cells density: 3,000 cells/mL
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4
- Preparation of glassware: All glassware used in testing was thoroughly scrubbed with non-phosphate detergent and rinsed with tap water. This was followed by a rinse with acetone, further rinses with deionized water, a rinse in 10% reagent grade hydrochloric acid, and thorough rinsing in deionized water, performed with ASTM Type I reagent grade water. Glassware was dried in an oven at 50 - 70°C and autoclaved for 20 minutes at 1.1 kg/cm2 and 121°C.

INOCULUM
- An algal inoculum was prepared from a 7-day old stock culture. Approximately 3 mL of the stock culture was diluted with 7 mL AAP/Si medium. Population density in the diluted sample of stock culture was determined with an electronic particle counter. The sample contained
705,480 cells/mL. A 0.213 mL volume of this sample was aseptically added to 50 mL of solution in each of the four replicate flasks per treatment.

GROWTH MEDIUM
- Preparation of medium: Synthetic AAP/Si medium was prepared by placing 202.4 mg of sodium silicate in a 1000 mL volumetric flask with approximately 900 mL Type I water. To this was added 1 mL of each of the macronutrient stock solutions and 1 mL of the macro/micronutrient stock solution, with mixing after each addition. The volume was brought to 1 liter and the pH was adjusted to 7.5 ± 0.1 with 0.1 N sodium hydroxide or hydrochloric acid. The medium was subsequently filtered through a 0.22 µ pore size membrane filter into a sterile container. Medium was prepared in advance of test initiation and stored under refrigeration prior to use.

WATER PARAMETERS
- Intervals of water quality measurement: The instantaneous temperature was manually read and recorded each day, while an automated system recorded temperature continuously.

OTHER TEST CONDITIONS
- Shaking speed: 100 oscillations per minute
- Light intensity: 4306 ± 646 lux (400 ± 60 foot-candles)
- Photoperiod: Continuous
- Flasks were randomly repositioned each working day (except weekends) to minimize spatial differences in the incubator.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell counts were made using the Coulter Counter on test days 3, 4 and 5. The Coulter Counter operates on the principle that cells are poor electrical conductors. The algal cells, suspended in an electrolyte, can be sized and counted by passing them through an aperture with a specific path of current flow. As cells pass through the aperture and displace a volume of electrolyte equal to their volume, the resistance changes, causing current and voltage changes which are translated into number and size of cells.
Three counts per replicate were made. All counts were multiplied by the appropriate conversion factors (for sample dilution and volume counted) to yield cells/mL. Samples of 0.2 to 2.0 mL in volume, depending upon the expected population density, were collected aseptically using an automatic micropipette with a sterile disposable tip. Sample dilutions were made with the electrolyte Isoton II as the diluent. Samples were not returned to the test flasks.

RANGE FINDING STUDY
- An initial range-finding test was conducted using five nominal concentrations from 0.0004 to 4 mg/L. The highest test concentration, 4 mg/L, is equivalent to the concentration required to be tested in a Tier 1 test, i.e. a concentration equivalent to the maximum label application rate as though the product was directly applied to the surface of a one-acre pond to a depth of six inches. The maximum label application rate for the test substance is 2.8 pounds per acre in two applications per year. The equivalent water concentration is 2.058 mg/L (Urban and Cook, 1986), or 4.1 mg/L for two applications.
- Result: Based upon the results of this range-finding test, subsequent range-finding testing was conducted, at lower concentrations. The results of the range-finding tests indicated that nominal concentrations from 0.250 to 8.0 µg/L would be appropriate for the definitive test.

Reference substance (positive control):

no

Key result

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

0.962 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

The raw data was used to derive the effect value based on growth rate. this calculation was performed in a separate assessment

Key result

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

4.155 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: The raw data was used to derive the effect value based on growth rate. this calculation was performed in a separate assessment

Remarks:

95% C.I.: 3.929 - 4.554 µg/L

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

1.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

95% C.L.: 1.12 - 1.75 µg/L

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

0.562 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

The result indicated that exposure to increasing concentrations of the test substance resulted in increasing inhibition of the population growth of Navicula pelliculosa. An overview of the results is provided in Table 2 at 'Any other information on results incl. tables'

BIOMASS
The biomass was determined by coulter counter on test days 3, 4 and 5. At the start of the test, the initial cell density was 3.0E+03 cells/mL. After 3 days (72-hours) of exposure, cell density in the negative control averaged approximately 3.29E+04 cells/mL and in the solvent control average approximately 2.86E+04 cells/mL. On day 3 (at 72-hour), the averaged algal density in the 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µg/L treatment levels were 2.14, 1.78, 2.63, 1.68, 1.33 and 1.09E+04 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the negative control averaged 1.84E+06 cells/mL and in the solvent control average approximately 1.60E+06 cells/mL. The 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µ/L treatment levels showed a 1.29, 1.18, 1.15, 0.47, 0.069 and 0.013E+06 cells/mL algal density, respectively, on day 5 (at 120-hour).

ECx AND NOEC
The effects of the test substance on mean standing crop on day 5, relative to the solvent control, ranged from 19.6% to 98.9% inhibition. As determined by weighted least squares nonlinear regression, the 5-day EC25 was 0.890 µg/L (95% confidence limits 0.650 - 1.22 µg/L) and the 5-day EC50 was 1.40 µg/L (95% confidence limits 1.12 - 1.75 µg/L). To determine the NOEC, ANOVA and Dunnett’s test were performed. These statistical analyses indicate that the mean standing crop values on day 5 in the four highest test concentrations were significantly different from the mean in the solvent control. The NOEC, based upon the selected test concentrations, results and calculations, was thus 0.562 µg/L.

Reported statistics and error estimates:

The data analysis is based upon the measured test concentrations. The mean of the results for the day 0 and day 5 analyses for each test concentration were used. The mean cell count values at test termination for each test concentration were expressed as a percentage relative to that in the solvent control.
To determine the EC25 and EC50 values and associated 95% confidence limits, weighted least squares nonlinear regression of the log of test concentration against the day 5 cell counts was performed. The NOEC was determined from an analysis of variance (ANOVA) and Dunnett’s test. Statistical analyses were performed using SAS Software (SAS Institute, Cary, NC). The level of significance was at a = 0.05.

Table 2. Percent inhibition¹, relative to solvent control, based upon mean standing crop, cells/mL, on day 5.

Concentration (µg/L)		Mean standing crop on day 5, cell/mL	Inhibition, %
Mean measured	Nominal
N.D.^2	Solvent control	1,601,000	---
N.D.	No- treatment control	1,838,200	-14.8
0.288	0.250	1,286,825	19.6
0.562	0.500	1,182,565	26.1
0.962	1.00	1,148,805**	28.2
1.87	2.00	469,660**	70.7
3.85	4.00	68,805**	95.7
8.02	8.00	17,908**	98.9

1 A negative percent inhibition indicates stimulation
2 N.D. = not detected; detection limit = 0.1 µg/L
** significantly different from solvent control at α = 0.05

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the 5-day NOEbC value was determined to be 0.562 µg/L, and the 5-day EbC50 value was determined to be 1.40 µg/L (with corresponding 95% confidence interval 1.12 - 1.75 µg/L). Aside from the study, the 5-day NOErC was determined to be 0.962 µg/L and ErC was determined to be 4.155 µg/L (with corresponding 95% confidence interval 3.929 - 4.554 µg/L).

Executive summary:

The influence of the test substance on the growth of the freshwater diatom, Navicula pelliculosa was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 Tier 2 and in compliance with GLP. The test substance was first dissolved in the solvent N,N-dimethylformamide (DMF) before further diluting in the test medium. The diatoms were exposed to measured concentrations of 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µg/L (measured by GC, nominal concentrations were 0.250, 0.500, 1.00, 2.00, 4.00 and 8.00 µg/L, respectively). In addition, a negative control and a solvent control (0.5 mL/L DMF) group were included in this study as well. All treatments were performed with four replicates. The test was carried out in an incubator with a temperature range of 24 ± 2 °C. Flasks were continuously shaken at a speed of 100 oscillations per minute. Illumination of 4306 ± 646 lux (400 ± 60 foot-candles) was provided by overhead cool-white continuous fluorescent lights. Flasks were randomly repositioned each working day (except weekends) to minimize spatial differences in the incubation. The pH of the test started from a range of 7.29 – 7.37 and ended with a range of 7.17 – 7.63. Biomass of the algae was determined by cell counts on days 3, 4 and 5 using a coulter counter.

At the start of the test, the initial cell density was 3.0E+03 cells/mL. After 3 days (72-hours) of exposure, cell density in the negative control averaged approximately 3.29E+04 cells/mL and approximately 2.86E+04 cells/mL in the solvent control. On day 3 (at 72-hour), the average algal density in the 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µ/L treatment levels were 2.14, 1.78, 2.63, 1.68, 1.33 and 1.09E+04 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the negative control averaged 1.84E+06 cells/mL and approximately 1.60E+06 cells/mL in the solvent control. The 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µ/L treatment levels showed algal densities of 1.29, 1.18, 1.15, 0.47, 0.069 and 0.018E+06 cells/mL, respectively, on day 5 (at 120-hour).

The algal growth at the test concentrations was inhibited in the range of 19.6% to 98.9%, based on biomass. As determined by weighted least squares non-linear regression, the 5-day EbC50 was 1.40 µg/L (95% confidence limits 1.12 - 1.75 µg/L). The NOEbC for growth inhibition was 0.562 µg/L. The study did not provide effect levels for growth rate and, therefore, they were calculated from the raw data by the registrant using an established process and commercial software. The 5-day NOErC was determined to be 0.962 µg/L and the 5-day ErC50 was determined to be 4.155 µg/L (with corresponding 95% confidence interval of 3.929 - 4.554 µg/L).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov 1992 to 25 Nov 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: U.S. EPA - FIFRA Guideline 123-2

Version / remarks:

Growth and Reproduction of Aquatic Plants, Tier 2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were analyzed for the actual concentration of the test substance present in the test solutions on day 0 and day 5. Samples for the day 0 analyses were portions of the test treatments used to begin the test. At test termination, the contents of the replicate flasks were combined, the pH was recorded, and the algae were removed by centrifugation at 3700 rpm for four minutes. The supernatants were then submitted for analyses.

Vehicle:

yes

Remarks:

N.N-dimethyformamide (DMF)

Details on test solutions:

A primary stock solution of 1.03 mg a.i./mL in the solvent N.N-dimethylformamide (DMF) was prepared by weighing 10.5 mg the test substance, to the nearest 0.1 mg, and diluting to 10.0 mL in a volumetric flask with DMF. The stock solution was mixed by inverting several times, resulting in a clear liquid. Other stock solutions (0.0005, 0.00125, 0.0025, 0.005, 0.01, 0.02 and 0.04 mg/mL in DMF) were prepared by serial dilution.
Test concentrations were prepared by adding 0.25 mL of the appropriate stock solution to MAA medium in 500 mL volumetric flasks to yield nominal test concentrations of 0.250, 0.625, 1.25, 2.50, 5.00, 10.0, and 20.0 µg/L, respectively. The no-treatment control contained medium only. A solvent control was prepared to contain the same amount of DMF that was added to each test treatment, i.e. 0.5 mL/L. After thorough mixing, 100 mL of the no-treatment control, solvent control and each test treatment were added to each of three replicate test vessels.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Source: Original culture from EPA Environmental Research Laboratory

METHOD OF CULTIVATION
- Medium: Synthetic marine algal assay nutrient medium
- Light intensity and source: Approximately 400 foot-candles (4306 lumens/m2), cool-white fluorescent illumination
- Photoperiod: 14 hours/10 hours (light/dark)
- Temperature: 20 ± 2°C
- Others: Flasks were manually shaken each working day. Transfers were made regularly into fresh medium to provide cultures in logarithmic phase of growth for toxicity test inoculations.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

5 d

Test temperature:

20 ± 2 °C

pH:

- Day 0: 8.02 - 8.08
- Day 5: 8.13 - 8.75

Salinity:

30‰

Nominal and measured concentrations:

- Nominal concentrations: negative control, solvent control, 0.250, 0.625, 1.25, 2.50, 5.00, 10.00 and 20.00 µg/L.
- Mean measured concentrations: N.D., N.D., 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µg/L, respectively (N.D. = not detected; detection limit = 0.112 µg/L). See Table 1 in "Any other information on materials and methods incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml chemically clean, autoclaved Erlenmeyer flasks
- Preparation of glassware: All glassware used in testing was thoroughly scrubbed with non-phosphate detergent and rinsed with tap water. This was followed by a rinse with acetone, further rinses with deionized water, a rinse in 10% reagent grade hydrochloric, acid, and thorough rinsing in deionized water. A final rinse was performed with ASTM Type I reagent grade water (Type I water). Glassware was dried in an oven at 50 - 70°C and autoclaved for 20 minutes at 1.1 kg/cm2 and 121°C.
- Type: Closed (foam plugs)
- Inoculum: An algal inoculum was prepared from a 7-day old stock culture. Population density in the stock culture was determined with an electronic particle counter. The sample contained 1,000,640 cells/mL. A 0.999 mL volume of this sample was aseptically added to 100 mL of solution in each of the three replicate flasks per treatment.
- Yield nominal Initial cells density: 10,000 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3
- No. of vessels per vehicle control: 3

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Marine algal assay nutrient medium (MAA) was prepared by adding nutrients to synthetic sea water. Synthetic sea water was prepared by adding approximately 32 grams of a commercial salt mix to a liter of ASTM Type I water. Salt mix was added with continuous stirring until the salinity was 30 parts per thousand when all the salt was dissolved. Salinity was measured using a refractometer. The synthetic sea water was then filter-sterilized by passing through a sterile 0.22 µ porosity membrane filter into a sterile container. Nutrients were added to the sterile synthetic seawater. For stock culture medium, 30 mL metal mix, 20 mL minor salt mix and 1.0 mL vitamin mix were added to 1 L (final volume) of synthetic sea water. Metal mix used for stock culture medium contains EDTA whereas that used for toxicity tests does not. To prepare medium for toxicity tests, 15 mL metal mix (without EDTA), 10 mL minor salt mix and 0.5 mL vitamin mix were added to 1 L (final volume) of synthetic sea water. The pH was adjusted to 8.1 ± 0.1 with 0.1 N sodium hydroxide or hydrochloric acid. Medium was prepared in advance of test initiation and stored under refrigeration prior to use.

OTHER TEST CONDITIONS
- Photoperiod and source: 14 hours/10 hours (light/dark), cool-white fluorescent
- Light intensity and location: 4306 ± 646 lux (400 ± 60 foot-candles), overhead
- The instantaneous temperature was manually read and recorded each day, while an automated system recorded temperature continuously.
- Flasks were randomly repositioned each working day (except weekends) to minimize spatial differences in the incubator.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell counts were made using the Coulter Counter on test days 3, 4 and 5. The Coulter Counter operates on the principle that cells are poor electrical conductors. The algal cells, suspended in an electrolyte, can be sized and counted by passing them through an aperture with a specific path of current flow. As cells pass through the aperture and displace a volume of electrolyte equal to their volume, the resistance changes, causing current and voltage changes which are translated into number and size of cells. Skeletonema costatum grows in short chains which easily pass through the aperture tube of the Coulter Counter. The Coulter Counter thus counts the algae as short chains rather than as individual cells.
Three counts per replicate were made. All counts were multiplied by the appropriate conversion factors (for sample dilution and volume counted) to yield cells/mL. Samples of 1.0 or 2.0 mL in volume, depending upon the expected population density, were collected aseptically using an automatic micropipette with a sterile disposable tip. Sample dilutions were made with the electrolyte Isoton II as the diluent. Samples were not returned to the test flasks.

TEST CONCENTRATIONS
- Range finding study: An initial range-finding test was conducted using five nominal concentrations from 0.0004 to 4 mg/L. The highest test concentration, 4 mg/L, is equivalent to the concentration required to be tested in a Tier 1 test, i.e. a concentration equivalent to the maximum label application rate as though the product was directly applied to the surface of a one-acre pond to a depth of six inches. The maximum label application rate for the test substance is 2.8 pounds per acre in two applications per year. The equivalent water concentration is 2.058 mg/L (Urban and Cook, 1986), or 4.1 mg/L for two applications.
- Subsequent range-finding study: Based upon the results of this range-finding test, subsequent range-finding testing was conducted at lower concentrations. The results of the range-finding tests indicated that nominal concentrations from 0.625 to 20 µg/L would be appropriate for the definitive test. In addition, a lower test concentration (0.250 µg/L) was added to ensure that the NOEC would be obtained.

Reference substance (positive control):

no

Key result

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

12.59 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: The calculation was performed aside from the study

Remarks:

95%C.I.: 12.22 - 13.08 µg/L

Key result

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

4.54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: The calculation was performed aside from the study

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

7.63 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other:

Remarks:

95% C.L.: 4.24 - 5.85 µg/L

Duration:

5 d

Dose descriptor:

NOEC

Effect conc.:

2.22 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

BIOMASS
The biomass was determined by coulter counter on test days 3, 4 and 5. At the start of the test, the initial cell density was 1.0E+04 cells/mL. After 3 days (72-hours) of exposure, cell density in the negative control averaged approximately 1.23E+05 cells/mL and in the solvent control average approximately 1.15E+05 cells/mL. On day 3 (at 72-hour), the averaged algal density in the 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L treatment levels were 1.14, 1.14, 1.21, 1.08, 0.71, 0.39 and 0.22E+05 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the negative control averaged 2.72E+05 cells/mL and in the solvent control average approximately 2.67E+05 cells/mL. The 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L treatment levels showed a 2.66, 2.60, 2.56, 2.68, 2.38, 0.73 and 0.27E+05 cells/mL algal density, respectively, on day 5 (at 120-hour). From the shapes of the growth curves, it was evident that exposure to the four lowest test concentrations of the test substance had no effect upon the population growth of Skeletonema costatum, while exposure to the three highest test concentrations resulted in increasing inhibition with increasing concentration. An overview of the results is provided in Table 2 at "Any other information on results incl. tables"

ECx AND NOEC
Effects of the test substance on mean standing crop on day 5, relative to the solvent control, ranged from 0.202% stimulation to 90.0% inhibition. As determined by weighted least squares nonlinear regression, the 5-day EC25 was 4.98 µg/L (95% confidence limits 4.24 - 5.85 µg/L) and the 5-day EC50 was 7.63 µg/L (95% confidence limits 6.86 - 8.49 µg/L). The result of Dunnett’s test indicated that the mean standing crop values on day 5 in the three highest test concentrations were significantly different from the mean in the solvent control. The NOEC, based upon the selected test concentrations, results and calculations, was thus 2.22 µg/L.

Reported statistics and error estimates:

To determine the EC25 and EC50 values and associated 95% confidence limits, weighted least squares nonlinear regression of the log of test concentration against the day 5 cell counts was performed. The NOEC was determined from an analysis of variance (ANOVA) and Dunnett’s test. Statistical analyses were performed using SAS Software. The level of significance was at a = 0.05.

Table 2. Percent inhibition¹, relative to solvent control, based upon mean standing crop, cells/mL, on day 5.

Concentration (µg/L)		Mean standing crop on day 5, cell/mL	Inhibition, %
Mean measured	Nominal
N.D.^2	Solvent control	267,033	---
N.D.	No- treatment control	271,507	-1.68
0.259	0.250	266,520	0.192
0.595	0.625	260,013	2.63
1.16	1.25	255,993	4.13
2.22	2.50	267,573	-0.202
4.54	5.00	238,427**	10.7
9.74	10.0	73,220**	72.6
18.8	20.0	26,823**	90.0

1 A negative percent inhibition indicates stimulation
2 N.D. = not detected; detection limit = 0.112 µg/L
** significantly different from solvent control at α = 0.05

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the 5-day NOEbC value was determined to be 2.22 µg/L, and the 5-day EbC50 value was determined to be 7.63 µg/L (with corresponding 95% confidence interval 6.86 - 8.49 µg/L). Aside from the study, the 5-day NOErC was determined to be 4.54 µg/L and ErC was determined to be 12.59 µg/L (with corresponding 95% confidence interval 12.22 - 13.08 µg/L).

Executive summary:

The influence of the test substance on the growth of the marine diatom species Skeletonema costatum was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 Tier 2 and in compliance with GLP. The test substance was first dissolved in the solvent N,N-dimethylformamide (DMF) before further diluting in the test medium (synthetic medium with 30‰ salinity). The diatoms were exposed to measured concentrations of 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L (measured by GC on day 0 and 5, nominal concentrations were 0.250, 0.625, 1.25, 2.50, 5.00, 10.0, 20.0 µ/L, respectively). In addition, a negative control and a solvent control (0.5 mL/L DMF) group were included in this study as well. All treatments were performed with three replicates. The test was carried out in an incubator with a temperature range of 20 ± 2 °C. Flasks were manually shaken once each working day. Illumination of 4306 ± 646 lux (400 ± 60 foot-candles) was provided by overhead cool-white fluorescent lights on a photoperiod of 14 hours light:10 hours dark. Flasks were randomly repositioned each working day (except weekends) to minimize spatial differences in the incubation. The pH of test medium at the start was in the range of 8.02 to 8.08 and changed to the range from 8.13 – 8.75 at the end of the test. Biomass of the algae was determined by cell counts on days 3, 4 and 5 using a coulter counter.

At the start of the test, the initial cell density was 1.0E+04 algal cells/mL for all replicates. After 3 days (72-hours) of exposure, cell density in the negative control averaged 1.23E+05 cells/mL and 1.15E+05 cells/mLinthe solvent control. On day 3, the average algal density in the 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L treatment levels was 1.14, 1.14, 1.21, 1.08, 0.71, 0.39 and 0.22E+05 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the negative control averaged 2.72E+05 cells/mL and  2.67E+05 cells/mL in the solvent control . The 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L treatment levels showed a cell density of 2.67, 2.60, 2.56, 2.68, 2.38, 0.73 and 0.27E+05 cells/mL, respectively, on day 5 (at 120 hours). From the shapes of the growth curves, it was evident that exposure to the four lowest test concentrations of the test substance had no effect upon the population growth of Skeletonema costatum, while exposure to the three highest test concentrations resulted in increasing inhibition with increasing concentration.

The effects of the test substance on the cell growth of the marine diatom ranged from 0.202% stimulation to 90.0% inhibition. As determined by weighted least squares nonlinear regression, the 5-day EbC50 was 7.63 µg/L (95% confidence limits 6.86 - 8.49 µg/L). The 5-day NOEC, based on biomass, was 2.22 µg/L. The study did not provide effect levels for growth rate, and these were calculated from the raw data by the registrant using an established process and commercial software. The 5-day NOErC was determined to be 4.54 µg/L and the ErC50 was determined to be 12.59 µg/L (with corresponding 95% confidence interval 12.22 - 13.08 µg/L).

Description of key information

All available data was assessed and the studies representing the worst-case effects are included here as key. The results can be considered worst-case and are selected for the CSA. Other studies are included as supporting information.

Freshwater 5-d NOErC = 0.962 µg/L, Navicula pelliculosa, EPA FIFRA TG 123 - 3, Hughes & Alexander 1992

Freshwater 5-d ErC50 = 4.155 µg/L (corresponding with 95% confidence limit of 3.929 - 4.554 µg/L), Navicula pelliculosa, EPA FIFRA TG 123 - 3, Hughes & Alexander 1992

Saltwater 5-d NOErC = 4.54 µg/L, Skeletonema costatum, EPA FIFRA TG 123 - 3, Hughes & Alexander 1993

Saltwater 5-d ErC50 = 12.59 µg/L (corresponding with 95% confidence limit of 12.22 - 13.08 µg/L), Skeletonema costatum, EPA FIFRA TG 123 - 3, Hughes & Alexander 1993

Key value for chemical safety assessment

EC50 for freshwater algae:

4.155 µg/L

EC50 for marine water algae:

12.59 µg/L

EC10 or NOEC for freshwater algae:

0.962 µg/L

EC10 or NOEC for marine water algae:

4.54 µg/L

Additional information

Freshwater  

The influence of the test substance on the growth of the freshwater diatom, Navicula pelliculosa was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 Tier 2 and in compliance with GLP. The test substance was first dissolved in the solvent N,N-dimethylformamide (DMF) before further diluting in the test medium. The diatoms were exposed to measured concentrations of 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µg/L (measured by GC, nominal concentrations were 0.250, 0.500, 1.00, 2.00, 4.00 and 8.00 µg/L, respectively). In addition, a negative control and a solvent control (0.5 mL/L DMF) group were included in this study as well. All treatments were performed with four replicates. The test was carried out in an incubator with a temperature range of 24 ± 2 °C. Flasks were continuously shaken at a speed of 100 oscillations per minute. Illumination of 4306 ± 646 lux (400 ± 60 foot-candles) was provided by overhead cool-white continuous fluorescent lights. Flasks were randomly repositioned each working day (except weekends) to minimize spatial differences in the incubation. The pH of the test started from a range of 7.29 – 7.37 and ended with a range of 7.17 – 7.63. Biomass of the algae was determined by cell counts on days 3, 4 and 5 using a coulter counter.

At the start of the test, the initial cell density was 3.0E+03 cells/mL. After 3 days (72-hours) of exposure, cell density in the negative control averaged approximately 3.29E+04 cells/mL and approximately 2.86E+04 cells/mL in the solvent control. On day 3 (at 72-hour), the average algal density in the 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µ/L treatment levels were 2.14, 1.78, 2.63, 1.68, 1.33 and 1.09E+04 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the negative control averaged 1.84E+06 cells/mL and approximately 1.60E+06 cells/mL in the solvent control. The 0.288, 0.562, 0.962, 1.87, 3.85 and 8.02 µ/L treatment levels showed algal densities of 1.29, 1.18, 1.15, 0.47, 0.069 and 0.018E+06 cells/mL, respectively, on day 5 (at 120-hour).

The algal growth at the test concentrations was inhibited in the range of 19.6% to 98.9%, based on biomass. As determined by weighted least squares non-linear regression, the 5-day EbC50 was 1.40 µg/L (95% confidence limits 1.12 - 1.75 µg/L). The NOEbC for growth inhibition was 0.562 µg/L. The study did not provide effect levels for growth rate and, therefore, they were calculated from the raw data by the registrant using an established process and commercial software. The 5-day NOErC was determined to be 0.962 µg/L and the 5-day ErC50 was determined to be 4.155 µg/L (with corresponding 95% confidence interval of 3.929 - 4.554 µg/L).

 

Marine water  

The influence of the test substance on the growth of the marine diatom species Skeletonema costatum was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 Tier 2 and in compliance with GLP. The test substance was first dissolved in the solvent N,N-dimethylformamide (DMF) before further diluting in the test medium (synthetic medium with 30‰ salinity). The diatoms were exposed to measured concentrations of 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L (measured by GC on day 0 and 5, nominal concentrations were 0.250, 0.625, 1.25, 2.50, 5.00, 10.0, 20.0 µ/L, respectively). In addition, a negative control and a solvent control (0.5 mL/L DMF) group were included in this study as well. All treatments were performed with three replicates. The test was carried out in an incubator with a temperature range of 20 ± 2 °C. Flasks were manually shaken once each working day. Illumination of 4306 ± 646 lux (400 ± 60 foot-candles) was provided by overhead cool-white fluorescent lights on a photoperiod of 14 hours light:10 hours dark. Flasks were randomly repositioned each working day (except weekends) to minimize spatial differences in the incubation. The pH of test medium at the start was in the range of 8.02 to 8.08 and changed to the range from 8.13 – 8.75 at the end of the test. Biomass of the algae was determined by cell counts on days 3, 4 and 5 using a coulter counter.

At the start of the test, the initial cell density was 1.0E+04 algal cells/mL for all replicates. After 3 days (72-hours) of exposure, cell density in the negative control averaged 1.23E+05 cells/mL and 1.15E+05 cells/mL in the solvent control. On day 3, the average algal density in the 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L treatment levels were 1.14, 1.14, 1.21, 1.08, 0.71, 0.39 and 0.22E+05 cells/mL, respectively. Following 5 days (120-hour) of exposure, cell biomass in the negative control averaged 2.72E+05 cells/mL and 2.67E+05 cells/mL in the solvent control. The 0.259, 0.595, 1.16, 2.22, 4.54, 9.74 and 18.8 µ/L treatment levels showed a cell density of 2.67, 2.60, 2.56, 2.68, 2.38, 0.73 and 0.27E+05 cells/mL, respectively, on day 5 (at 120 hours). From the shapes of the growth curves, it was evident that exposure to the four lowest test concentrations of the test substance had no effect upon the population growth of Skeletonema costatum, while exposure to the three highest test concentrations resulted in increasing inhibition with increasing concentration.

The effects of the test substance on the cell growth of the marine diatom ranged from 0.202% stimulation to 90.0% inhibition. As determined by weighted least squares nonlinear regression, the 5-day EbC50 was 7.63 µg/L (95% confidence limits 6.86 - 8.49 µg/L). The 5-day NOEC, based on biomass, was 2.22 µg/L. The study did not provide effect levels for growth rate, and these were calculated from the raw data by the registrant using an established process and commercial software. The 5-day NOErC was determined to be 4.54 µg/L and the ErC50 was determined to be 12.59 µg/L (with corresponding 95% confidence interval 12.22 - 13.08 µg/L).