1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Aug - 01 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1025 (Bivalve Acute Toxicity (shell deposition test))

Version / remarks:

draft version 1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.31, 0.65, 1.3, 2.5 and 5.0 mg active ingredient (a.i.)/L
- Sampling method: Samples were taken from the single test chamber in each treatment and control group three days prior to the start of exposure to confirm concentrations after conditioning the diluter system for two days. Test water samples also were collected from each test chamber at the beginning of the test and at 48 and 96 h (± 1 h) to measure concentrations of the test substance. The samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.

Vehicle:

yes

Remarks:

dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutione were prepared once for each of the five concentrations tested; detailed description in section " Any other information on material and methods".
- Controls: yes (dilution water and algae feed suspension)
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): The concentration of DMF in the solvent control and all treatment groups was 0.1 mL/L.

Test organisms (species):

other aquatic mollusc: Crassostrea virginica

Details on test organisms:

TEST ORGANISM
- Common name: eastern oyster
- Source: Circle C Oyster Ranch, Dameron, Maryland, USA
- Age of parental stock (mean and range, SD): no data
- Feeding during test: yes
- Food type: suspension of marine microalgae (Phyto-Feast from Reed Mariculture, Inc. of Campbell, California) provided through peristaltic pumps (Cole-Parmer Instrument Company of Chicago, Illinois)
- Amount: a nominal rate of approximately 5.8 x 10E+09 cells/oyster/day.
- Frequency: continuously

ACCLIMATION
- Acclimation period: 12 d
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: same as test
- Feeding frequency: approximately 2.9 x 10E+9 cells/oyster/day
- Health during acclimation (any mortality observed): During the 7 d period immediately preceding the test, the oysters in the lot used for the test showed no signs of disease or stress and there were no mortalities.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Mean: 32.9 ± 1.8 mm, range: 30.2 - 36.1 mm

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

0 h: 20.3 - 20.5 °C
96 h: 20.0 - 20.5 °C
Dilution water: 19.0 - 21.2 °C

pH:

0 h: 7.8
48 h: 7.9
96 h: 7.8 - 7.9
Dilution water: 7.8 (Mean of 4 week period immediately preceding the test)

Dissolved oxygen:

0 h: 7.9 - 8.1 mg/L
24 h: 6.8 - 7.1 mg/L
48 h: 6.8 - 7.6 mg/L
72 h: 6.6 - 7.5 mg/L
96 h: 6.2 - 7.3 mg/L
Dilution water: ≥ 6.5 mg/L (≥ 81% of saturation)

Salinity:

0 h and 96 h: 20 parts per thousand (‰)
Dilution water: 20 to 21 ‰

Nominal and measured concentrations:

Control, Solvent control, 0.31, 0.65, 1.3, 2.5 and 5.0 mg a.i./L (nominal)
< LOQ, < LOQ, 0.31, 0.66, 1.3, 2.6 and 5.1 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
Test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 54 L glass aquaria filled with approx. 27 L of test water. The depth of the test water in a representative chamber was 14.8 cm. The test chambers were indiscriminately positioned in a temperature-controlled environmental chamber
- Aeration: no aeration during the environmental conditions; Dilution water was aerated using a spray nozzles
- Type of flow-through (e.g. peristaltic or proportional diluter): Syringe pumps (Harvard Apparatus, Holliston, Massachusetts)
- Renewal rate of test solution (frequency/flow rate): each test chamber received approx. 19 volume additions of solution every 24 h
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered natural seawater diluted to a salinity of 20‰ with freshwater collected from a well.
- Total organic carbon (dilution water): < 1 mg C/L (Taken monthly)
- Metals: <0.0002 - 12400 µg/L
- Pesticides and Organics: < 0.0081 - < 4.8 µg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: quality of test water measured after 0 h (pH, dissolved oxygen, temperature, salinity); 24 h (dissolved oxygen); 48 h (dissolved oxygen, pH); 72 h (dissolved oxygen); 96 h (pH, dissolved oxygen, temp, salinity)

OTHER TEST CONDITIONS
- Photoperiod: 16 h of light and 8 h of darkness. A 30 min transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity.
- Light intensity: fluorescent tubes that emit wavelengths similar to natural sunlight (118 lux at the surface of the water of one representative test chamber).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Any oysters having open shells and not responding to gentle prodding were considered dead and removed. The numbers of individuals exhibiting signs of toxicity, or abnormal behavior or visible abnormalities such as gaping of the shell, excessive mucus production, spawning, or abnormal feeding activity, also were evaluated. Observations were made approximately 6, 24, 48, 72 and 96 h after test initiation. At the end of the test, shell deposition was recorded for each oyster by measuring the length of the longest finger of new shell to the nearest 0.1 mm using calipers.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: Concentrations of the definitive test were based on the results of a non-GLP range-finding test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: shell deposition

Remarks on result:

other: 95 % confidence interval

Remarks:

1.9 - 2.4 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: shell deposition

Details on results:

- Behavioural abnormalities: No mortalities occurred among oysters in any treatment group during the test, and all oysters appeared normal throughout the test.
- Observations on body length and weight: Inhibition of shell growth in the 0.31, 0.66, 1.3, 2.6 and 5.1 mg a.i./L treatment groups was -22, -3, 17, 80 and 99%, respectively.
- Other biological observations: Statistically significant decrease was observed in shell deposition at 2.6 and 5.1 mg a.i./L treatment groups in comparison to the control (p ≤ 0.05).
- Mortality of control: No mortalities occurred among oysters in any control.
- Any observations (e.g. precipitation): The test solutions appeared clear and colorless, with no evidence of precipitation observed during the test, in the negative control, solvent control, 0.31, 0.65, 1.3 and 2.5 mg a.i./L test chambers, and in the diluter mixing chambers that delivered test solution for these concentrations. Test solutions in the 5.0 mg a.i./L test chambers appeared clear and colorless with no precipitates observed at test initiation and termination. The solution in the diluter mixing chamber that delivered test solution for the 5.0 mg a.i./L test concentration also appeared clear and colorless, but white precipitate was noted on the sides of the mixing chamber.
- Other: Biological and analytical results are summarized in Table 1 and 2 within the section "Any other information on results incl. tables"

Results with reference substance (positive control):

No reference substance was tested.

Reported statistics and error estimates:

The shell deposition data from the negative control and solvent control groups were compared using a t-test. Since no significant differences were detected between the two control groups (p > 0.05), the control data were pooled for comparison of growth inhibition in the treatment groups. The EC50 value for shell deposition was calculated using linear interpolation. The shell deposition data were evaluated for normality and homogeneity of variance (p = 0.01) using the Chi-Square and Levene’s tests, respectively. Since the assumption of homogeneity was not met, the data in the treatment groups were compared to the pooled control data using the Wilcoxon’s Rank Sum test with a Bonferroni adjustment to identify any significant differences (p = 0.05). The no-observed-effect concentration (NOEC) was determined from the statistical analyses of the data and an assessment of the concentration-response pattern. Statistical analyses were conducted using a personal computer with TOXSTAT software.

The negative control for the shell deposition and shell growth data were compared with the solvent control and no statistically significant differences were found at the 95% level of confidence, respectively. Therefore, the control data were pooled for comparison with the treatment groups, respectively.

Analytical results:

Measured concentrations of the samples ranged from approximately 95 to 109% of nominal. Measured concentrations of the samples ranged from approximately 97 to 113% of nominal. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 0.31, 0.66, 1.3, 2.6 and 5.1 mg a.i./L, representing 100, 102, 100, 104 and 102% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Table 1: Measured Concentrations of the test substance test medium

Nominal Test Concentration (mg a.i./L)	Sample Number (149A-250-)	Sampling Time (Days)	Measured Concentration (mg a.i./L)1	Percent of Nominal2	Mean Measured Concentration (mg a.i./L)	Mean Measured Percent of Nominal
Negative Control	1	0	< LOQ	--	--	--
	8	2	< LOQ	--
	15	4	< LOQ
Solvent Control	2	0	< LOQ	--	--	--
	9	2	< LOQ	--
	16	4	< LOQ
0.31	3	0	0.307	99.0	0.31 ± 0.0142 CV = 4.54%	100
	10	2	0.301	96.9
	17	4	0.328	106
0.65	4	0	0.665	102	0.66 ± 0.0191 CV = 2.90%	102
	11	2	0.638	98.2
	18	4	0.675	104
1.3	5	0	1.36	105	1.3 ± 0.0611 CV = 4.54%	100
	12	2	1.28	98.5
	19	4	1.40	108
2.5	6	0	2.75	110	2.6 ± 0.150 CV = 5.80%	104
	13	2	2.58	103
	20A3	4	2.45	97.9
5.0	7	0	5.64	113	5.1 ± 0.428 CV = 8.31%	102
	14	2	4.88	97.6
	21A3	4	4.92	98.3

1 The limit of quantitation (LOQ) was 0.100 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10.0).

2 Results were generated using Excel 2010 in the full precision mode. Manual calculations may differ slightly.

3 Samples were reanalyzed

Table 2: Mean Shell Deposition and Shell Growth Inhibition at 96 h

Mean Measured Concentration (mg a.i./L)	Mean Shell Deposition ± Standard Deviation (mm)	Shell Growth Inhibition 1,2 (%)
Negative Control	2.8 ± 1.45	--
Solvent Control	2.4 ± 1.14	--
Pooled Control	2.6 ± 1.30	--
0.31	3.4 ± 1.68	-22
0.66	2.9 ± 1.28	-3
1.3	2.3 ± 1.01	17
2.6	0.55 ± 0.78*	80
5.1	0.04 ± 0.12*	99

1 Shell growth inhibition was calculated relative to the pooled control.

2 96 hour EC50 (95% confidence interval) = 2.2 mg a.i./L (1.9 – 2.4 mg a.i./L).

* Statistically significant difference from the pooled control using the Wilcoxon’s Rank Sum test.

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 July 2013 - 29 Nov 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 13 April, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, solvent control, 47.9, 81.4, 138, 235 and 400 µg a.i./L
- Sampling method: water samples were taken on experimental day 0 (test initiation), day 1 and day 2 (test termination)
- Sample storage conditions before analysis: freezer

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution Series
- Controls: Untreated dilution water (blank) control, Solvent control
- Chemical name of vehicle: Dimethylformamide (DMF)
- Concentration of vehicle in test medium: 100µL DMF/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Genotype: type B (according to BAIRD, D.J. et al. (1991) A Comparative Study of Genotype Sensitivity to Acute Toxic Stress Using Clones of Daphnia magna Strauss. Ecotoxicological and Environmental Safety 21, 257-265)
- Source: Bayer CropScience laboratory breeding stock (in-house cultures), originally obtained from Bundesgesundheitsamt, Germany in 1982 as EEC-ringtesting participant
- Age of parental stock: Third (or later) brood of coeval parent daphnids (20-28 days ± 12 hours old)
- Age at test initiation: First instar, less than 24 hours old neonates

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Repeated sieving of a breeding-culture of defined age, less than 24h before test initiation.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

214 mg/L CaCO3

Test temperature:

20.2 - 21.6 °C (control)
20.6 - 22.0 °C (400 µg a.i./L)

pH:

7.7 - 7.9 (control)
7.8 - 8.0 (solvent control)
7.8 - 8.0 (test concentrations)

Dissolved oxygen:

8.5 - 8.7 mg O2/L (control)
8.6 - 8.7 mg O2/L (solvent control)
8.6 - 8.8 mg O2/L(test concentrations)

Conductivity:

621 μS/cm (day 0) , 628 μS/cm (day 1) (Dilution water)

Nominal and measured concentrations:

control, solvent control, 47.9, 81.4, 138, 235 and 400 μg a.i./L (nominal)
for measured values see Tables 1and 2 in section "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers (DIN 12332)
- Type: closed (covered with transparent glass plates)
- Material, size, headspace, fill volume: 100 mL glass beakers (DIN 12332), filled with 50 mL of the test solution
- Renewal rate of test solution: renewed after 24h of exposure
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water is fully defined, artificial water (type “Elendt M7"). Batch preparation (stock volume: 1000 L): Analytical grade mineral salts, dissolved in deionised water with a conductivity below 10μS/cm. Immediately before use in a study, the vitamin components were added, using a separately deep frozen stored stock solution.
- Alkalinity: 53 mg/L CaCO3
- Conductivity: 621 μS/cm (day 0) , 628 μS/cm (day 1)
- Intervals of water quality measurement: water samples were taken on experimental day 0 (test initiation), day 1 and day 2 (test termination)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: max. 1200 lux

EFFECT PARAMETERS MEASURED: Cumulative mortality (observed as immobility) and Behavioral Observations at 24 and 48 h

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: Non-GLP assay, using nominal test concentrations of 200 μg a.s./L and 400 μg a.s./L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

247 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 226 - 271 µg a.i./L

Results with reference substance (positive control):

- Results with reference substance valid? yes; Acute non-GLP reference testing, performed at least twice a year, using potassium dichromate
- Dose-response test: yes
- Test concentrations: control, 0.56, 0.75, 1.00, 1.33 and 1.78 mg K2Cr2O7/L
- EC50: 0.93 mg/L (range of 0.6–2.1 mg/L (OECD ringtesting, published with OECD Guideline 202 (2004))

Reported statistics and error estimates:

24 h: EC50 range = 270 - 334 µg a.i./L (lower-upper 95% CI)
48 h: EC50 range = 226 - 271 µg a.i./L (lower-upper 95% CI)

Analytical Results:

The measured amounts of the item in the freshly prepared test solutions at the start of each renewal interval revealed recoveries between 86% and 116% (mean: 108%) of nominal concentrations. The corresponding concentrations of the aged test solutions at the end of each 24-hour exposure period ranged between 83% and 113% (mean: 101%) of nominal. No contaminations of the test item were detected in samples from untreated water control.

Biological Results:

No immobility or other effects on behaviour occurred in untreated control, solvent control or treatment groups less than or equal to 81.4 μg a.s./L within the 48 hours of exposure. Only one minor sublethal observation in one test replicate of 138 μg a.s./L was made.

Table 1: Number and percentage of immobilised daphnids

nominal test concentration (μg a.s./L)	exposed daphnids (=100%)	immobilised daphnids
		24 h		48 h
		n	%	n	%
control	30	0	0.0	0	0.0
solvent control	30	0	0.0	0	0.0
47.9	30	0	0.0	0	0.0
81.4	30	0	0.0	0	0.0
138	30	0	0.0	0	0.0
235	30	4	13.3	11	36.7
400	30	26	86.7	30	100

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Mar 2014 - 02 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1035 (Mysid Acute Toxicity Test)

Version / remarks:

1996 / Public Draft

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, solvent control, 0.56, 1.1, 2.3, 4.5 and 9.0 mg a.i./L
- Sampling method: Test water samples were collected from one replicate test chamber of each treatment and control group two days prior to the start of exposure. Test water samples also were collected from one replicate test chamber in each treatment and control group at the beginning of the test and at 48 and 96 hours (± 1 hour) . The samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individual stock solutions were prepared for each of the five concentrations tested. Three primary stock solutions were prepared at volumes of 224, 128 and 128-mL by mixing calculated amount of test substance into DMF at nominal concentrations of 23, 45 and 90 mg a.i./mL, respectively. The 45 and 90 mg a.i./mL stock solutions were used for the two highest concentrations tested. The 23 mg a.i./mL was used to prepare two secondary stock solutions (128 mL each), and also served as the third highest concentration tested. The secondary stock solutions were prepared in DMF at nominal concentrations of 5.6 and 11 mg a.i./mL by proportional dilution (31.17 and 61.22 mL, respectively) of the 23 mg a.i./mL primary stock. Fresh aliquots of each stock were placed on the delivery system pumps every one to three days during the test. During the exposure period, the stock solutions were pumped into the diluter mixing chambers assigned to the treatment groups at a target rate of 12.50 μL/minute and were mixed with dilution water in the mixing chambers, delivered at a target rate of 125 mL/minute to achieve the desired nominal test concentrations.
- Controls: blank control, solvent control
- Chemical name of vehicle: DMF
- Concentration of vehicle in test medium: 0.1 mL/L

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: saltwater mysid
- Source: in-house cultures Wildlife International
- Age: juveniles (< 24 h old)
- Feeding during test: yes
- Food type: nutrient-enriched brine shrimp supplied by INVE Aquaculture, Salt Lake City, Utah, United States
- Frequency: twice daily (with exception of test termination)

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions: same as test
- Type and amount of food: live brine shrimp nauplii (Artemia sp.) daily, supplied by INVE Aquaculture, Salt Lake City, Utah. The brine shrimp periodically were enriched with a nutrient enrichment (A1 DHA Selco from INVE Aquaculture, Phichit, Thailand).
- Feeding frequency: daily
- Health during acclimation: no signs of disease or stress.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24.6 °C (control, at 0 h)
24.5 - 24.6 °C (solvent control, at 0h)
24.6 - 24.7 °C (test concentrations)

pH:

7.8 - 7.9 (control, 0 - 48 h range)
7.8 - 7.9 (solvent control, 0 - 48 h range)
7.8 - 7.9 (test concentrations)

Dissolved oxygen:

7.4 mg O2/L (control, 0 - 48 h)
7.4 mg O2/L (solvent control, 0 - 48 h)
7.1 - 7.4 mg O2/L (test concentrations)

Salinity:

20‰

Nominal and measured concentrations:

control, solvent control, 0.56, 1.1, 2.3, 4.5 and 9.0 mg a.i./L (nominal)
< LOQ, < LOQ, 0.55, 1.1, 2.2, 4.9 and 9.8 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 25-L Teflon®-lined stainless steel aquaria filled with approximately 15 L of test water; depth of test water: 17.5 cm; test chamber contains one test department (glass culture dish, diameter = 12 cm, height = 19 cm, water depth = 10 cm)
- Type of flow-through: continuous-flow diluter
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater collected at Indian River Inlet, Delaware, United States. The seawater was pumped into a 5000-gallon holding tank and ozonated, before being filtered through a sand filter and delivered to 37,800-L holding tank. The water in the holding tank was aerated using spray nozzles. The salinity of the filtered seawater was diluted to approximately 20‰ (parts per thousand) with freshwater from a well on the Wildlife International site in the holding tank. Prior to use, the water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: < 1 mg C/L
- Salinity: 20 ‰
- Culture medium different from test medium: no
- Intervals of water quality measurement: two days prior to the start of exposure, at the beginning of the test and at 48 and 96 hours (± 1 hour)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness with 30 min transition period
- Light intensity: 404 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality and other signs of toxicity at approx. 4.5, 24, 48, 72 and 96 hours after test initiation

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY: yes

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 9.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Mortality of control: 0%

Mean recovery of test item concentration was 98 - 109%.

Table 1: Cumulative Mortality and Observations (AN = appear normal; No. Dead = Cumulative number of dead mysids):

Mean Measured Concentration (mg a.i./L)	Rep.	No. Exposed	No. Dead	Observations	No. Dead	Observations	No. Dead	Observations
Negative Control	A	10	0	10 AN	0	10 AN	0	10 AN
	B	10	0	10 AN	0	10 AN	0	10 AN
Solvent Control	A	10	0	10 AN	0	10 AN	0	10 AN
	B	10	0	10 AN	0	10 AN	0	10 AN
0.55	A	10	0	10 AN	0	10 AN	0	10 AN
	B	10	0	10 AN	0	10 AN	0	10 AN
1.1	A	10	0	10 AN	0	10 AN	0	10 AN
	B	10	0	10 AN	0	10 AN	0	10 AN
2.2	A	10	0	10 AN	0	10 AN	0	10 AN
	B	10	0	10 AN	0	10 AN	0	10 AN
4.9	A	10	0	10 AN	0	10 AN	0	10 AN
	B	10	0	10 AN	0	10 AN	0	10 AN
9.8	A	10	0	10 AN	0	10 AN	2	8 AN
	B	10	0	10 AN	0	10 AN	2	8 AN

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Aug - 08 Sep 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

public draft 1996 (modified)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 Nousan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 41.9, 71.2, 121, 206 and 350 μg a.s./L (nominal)
- Sampling method: sampled freshly prepared test item concentrations and aged test item concentrations from all replicates of a treatment group and control group
- Sample storage conditions before analysis: below -18 °C in a freezer until measurement occurred

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dilution Series; stock solution A was stirred for 35 min by a magnetic stirrer; Further stock solutions (B to E) were prepared based on stock solution A. The aqueous exposure concentrations were prepared as sub-dilutions of the corresponding solvent stock solution (A to E) and stirred for 85 min by a magnetic stirrer (detailed information is presented in table 1 "Any other information on material and methods incl tables")
- Controls: Untreated dilution water (blank) control, Solvent control
- Chemical name of vehicle: Dimethylformamide (DMF)
- Concentration of vehicle in test medium: 100 µL DMF/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: genotype No. 2 by Dr. Bradley, University of Sheffield, Department of Zoology, report of February 3, 1988. This clone was renamed later as "type B" according to BAIRD, D.J. et al. (1991)
- Source: Bundesgesundheitsamt, Germany; obtained in 1982 as EEC-ringtesting participant
- Age of parental stock (mean and range, SD): 20 - 28 d ± 12 h old
- Feeding during test: no
- Food type: During holding with living cells of the green algae Desmodesmus subspicatus in aqueous suspension
- Frequency: three times per week
- Age at test initiation: The first instars used in the test were less than 24 h old. They descend from at least the third (or later) brood of coeval parent daphnids (20-28 d ± 12 h old)

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Repeated sieving of a breeding-culture of defined age, less than 24 h before test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

231.4 mg/L CaCO3

Test temperature:

21.9 - 20.8 °C (control)
21.1 - 22.1 °C (350 µg/L)

pH:

6.8 - 6.6 (control)
6.8 (solvent control)
6.9 - 7.2 (test item concentrations)

Dissolved oxygen:

8.4 - 8.5 mg/L (control)
8.4 - 8.6 mg/L (solvent control)
8.4 - 8.8 mg/L (test item concentarrions)

Conductivity:

592 μS/cm

Nominal and measured concentrations:

control, solvent control, 41.9, 71.2, 121, 206 and 350 μg a.s./L (nominal)
<0.125, < 0.125, 36.7 - 40.2, 60.9 - 65.2, 105-108, 179-192 and 295-331 µg a.s. /L (measured concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with transparent glass plates)
- Material, size, headspace, fill volume: 100 mL glass beakers (DIN 12332), filled with 50 mL of the test solution (10 mL test solution per daphnid), corresponding to a fluid level of approximately 3 cm height
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water is fully defined, artificial water (type “Elendt M7"). Batch preparation (stock volume: 1000 L): Analytical grade mineral salts, dissolved in deionised water with a conductivity below 10 μS/cm. Immediately before use in a study, the vitamin components were added, using a separately deep frozen stored stock solution.
- Alkalinity: 53 mg/L CaCO3, equivalent to an acid-binding-capacity of 1 mL 0.1 N HCl
- Culture medium different from test medium: no
- Intervals of water quality measurement: Residue-analysis of artificially prepared fish testing water (inorganic contaminants, pesticide residue contaminants, organochlorine contaminants), performed at least two times per year for artificially prepared fish testing water.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light, 8 h dark
- Light intensity: < 1200 lux, 5400 K (cool white), LED (light-emitting-diode) panel

EFFECT PARAMETERS MEASURED: mobility after 24 and 48 h: defined as animals with swimming movements within approximately 15 sec after gentle agitation of the test vessel; sublethal effects: visual comparison of untreated control animals and treated animals, performed after 24 and 48 h of exposure.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: yes, non-GLP
- Results used to determine the conditions for the definitive study: Non-GLP assay, using nominal test concentrations of 150, 195, 253.5 and 329.6 μg/L to verify suitability of the chosen concentration range for EC50 calculation.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

173.3 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 147.2 - 199.9 µg a.s./L

Details on results:

- Behavioural abnormalities: 121 and 206 µg a.s./L: frequency of antennae movements distinctly decreased
- Mortality of control: No immobility or other effects on behaviour were observed in the control.
- Others: Biological and analytical results are summarized within the tables 1 and 2 in section "Any other information on results incl. tables"

Results with reference substance (positive control):

- Dose-response test: yes, 0.56, 0.75, 1.00, 1.33 and 1.78 mg/L
- ECx: 24 h EC50 of 0.70 mg/L

Reported statistics and error estimates:

Statistical method: Dose-response functions in aquatic toxicity testing and the Weibull model, fitted by an iterative weighted linear regression according to the Maximum Likelihood principle
As the study covered pure water control and an additional solvent control, adequate analysis for detection of statistically significant differences between controls was performed (Fisher’s Exact Binomial Test for pairwise comparisons on a 5% level of significance ,two-sided probability)
Software used: ToxRat-Professional, Vers.3.2.1 of the ToxRat Solutions GmbH, Germany

Analytical Results:

As these measured concentrations ranged well within the recommended range of 80 – 120 % of nominal, all reported results are based on nominal concentrations of the test item in the test solutions.

Table 1: Biological Results

nominal test concentration (μg a.s/L)	exposed daphnids (=100%)	immobilised daphnids
		24 h		48 h
		N	%	n	%
Control	30	0	0	0	0
solvent control*)	30	1	3.3	1	3.3
41.9	30	0	0	2	6.7
71.2	30	0	0	5	16.7
121	30	2	6.7	7	23.3
206	30	13	43.3	15	50.0
350	30	22	73.3	30	100

*) 0.1 mL dimethylformamide/L test solution)

Table 2: Analysed concentrations in test solutions

nominal test concentrations (μg a.s/L)	analysed concentrations of the freshly prepared solutions		analysed concentrations of the aged solutions after 48 h.
	μg a.s./L	% of nominal	μg a.s./L	% of nominal
control	<0.125	---	<0.125	---
solvent control	<0.125	---	<0.125	---
41.9	40.2	96	36.7	88
71.2	65.2	92	60.9	86
121	108.0	89	105	87
206	192	93	179	87
350	331	94	295	84
	mean: 92.8% of nominal		mean: 86.4% of nominal

 

Validity criteria fulfilled:

yes

Description of key information

Freshwater:

EC50 (48 h) = 0.1733 mg a.s./L (M-566522-02-1, Daphnia magna, nominal, OECD 202, key study)

Marine water:

EC50 (96 h) = 2.2 mg a.s./L (M-507732-01-1, Crassostrea virginica, mean measured, OPPTS 850.1025, key study)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.173 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

2.2 mg/L

Additional information

Four experimental studies are available on the short term toxicity of the test item (M-566522-02-1, M-502273-01-1, M-507732-01-1 and M-503278-01-1 and M-503278-01-1). Two studies were conducted with the freshwater species Daphnia magna and two with the marine species Crassostrea virginica and Americamysis bahia, respectively. The studies were performed according to OECD guideline 202 (in case of Daphnia magna), according to OPPTS 850.1025 (in case of Crassostrea virginica) and OPPTS 850.1035 (in case of Americamysis bahia). The nominal concentrations were verified by analytical monitoring. All studies were performed according to GLP. Due to the moderate water solubility the test items were initially dissolved in dimethylformamide to prepare test solutions.

In the key study (M-566522-02-1) Daphnia magna was exposed over a period of 48 h to nominal concentrations of 41.9, 71.2, 121, 206 and 350 µg a.s./L without feeding in a static system. The measured amounts of the test item in the freshly prepared test solutions at test initiation revealed recoveries between 89% and 96% (mean: 92.8%) of nominal concentrations. The corresponding concentrations of the aged test solutions at the end of the 48-hour exposure period ranged between 84% and 88% (mean: 86.4%) of nominal, demonstrating stability in the test system. Thus, all reported results are based on nominal concentrations. No immobility or other effects on behaviour were observed in the untreated control within 48 h of exposure. In the solvent control, one daphnid was immobile after 24 h but there were no other effects on behaviour observed in this group. After 48 h of exposure, 6.7% of daphnids were immobile in the group with test item concentration of 41.9 µg a.s./L and 100% (n = 30) were immobile at the highest test item concentration of 350 µg a.s./L. The EC50 (48 h) was calculated to be 173.3 µg a.s./L (95% CL: 147.2 – 199.9 µg a.s./L) based on the nominal test concentrations.

In the second study (M-502273-01-1) Daphnia magna were exposed under static conditions over a period of 48 h to nominal concentrations of 47.9, 81.4, 138, 235 and 400 µg a.s./L without feeding. The measured amounts of the test item in the freshly prepared test solutions at the start of each renewal interval revealed recoveries between 86% and 116% (mean: 108%) of nominal concentrations. The corresponding concentrations of the aged test solutions at the end of each 24-hour exposure period ranged between 83% and 113% (mean: 101%) of nominal. As the measured concentrations were within the recommended range of 80 – 120% of nominal, all reported results are based on nominal concentrations of the test item. The EC50 (48 h) was determined to be 247 µg a.s./L (95% Cl: nominally 226 – 271 µg a.s./L) based on the nominal concentrations.

The marine species eastern oyster (Crassostrea virginica, M-507732-01-1) were exposed in a flow-through system over a period of 96 h to a geometric series of five nominal (mean measured) concentrations of 0.31 (0.31), 0.65 (0.66), 1.3 (1.3), 2.5 (2.6) and 5.0 (5.1) mg a.s./L, respectively. Shell deposition, mortality and sublethal behavioural effects were used to determine the effect concentrations. Percent inhibition of shell growth in each treatment group was calculated relative to the pooled control data. There were no mortalities or clinical signs of toxicity observed at any concentration tested. The EC50 (96 h) for shell deposition was 2.2 mg a.s./L, the NOEC (96 h) was 1.3 mg a.s./L. The effect concentrations were based on the arithmetic mean measured concentrations.

In a second study the marine species Americamysis bahia (M-503278-01-1) was exposed in a flow through system over a period of 96 h to a geometric series of five nominal concentrations of 0.56, 1.1, 2.3, 4.5 and 9.0 mg a.s./L. Test item concentrations were analytically verified for each treatment and control group at the beginning and the end of the test. The mean measured test concentrations for this study were 0.55, 1.1, 2.2, 4.9 and 9.8 mg a.s./L, representing 98, 100, 96, 109 and 109% of nominal concentrations, respectively. Percent mortality in the 4.9 and 9.8 mg a.s./L treatment groups at test termination was 5 and 70%, respectively. Signs of toxicity observed among the mysids were only observed at 4.9 and 9.8 mg a.s./L at test termination included surfacing, erratic swimming, and lethargy. The LC50 (96 h) was 8.3 mg a.s./L, (95% CL: 7.0 to 10.0 mg a.s./L). The NOEC (96 h) was 2.2 mg a.s./L. All effect concentrations were based on the mean measured concentrations.