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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Feb 2014 - 02 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

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, 13.3, 24.0, 43.2, 77.8, 140 µg a.i./L
- Sampling method: Samples were taken on experimental days 0, 9 and 19 (freshly prepared media) and days 2, 12 and 21 (aged media), respectively
- Sample storage conditions before analysis: freezer

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: dilution series; for more detailed information on the preparation of test solution see table 1 in section "Any other information on materials and methods"
- Controls: blank control, solvent control
- Chemical name of vehicle: DMF
- Concentration of vehicle in test medium: 100μL/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
- Feeding during test: yes
- Food type: Concentrated algae suspension in daphnid dilution water
- Amount: 0.1 - 0.2 mg TOC per test vessel with 100 mL, corresponding to 1 x 108 cells/L
- Frequency: daily

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:

21 d

Hardness:

12 - 14 °dH (control)
12 - 14 °dH (solvent control)
12 - 14 °dH (140 µg a.i./L)

Test temperature:

range of freshly prepared and aged solutions:
19.9 - 20.8 °C (control)
19.9 - 20.9 °C (solvent control)
19.8 - 21.1 °C (test concentrations)

pH:

range of freshly prepared and aged solutions:
7.9 - 8.1 (control)
7.5 - 8.1 (solvent control)
7.5 - 8.5 (test concentrations)

Dissolved oxygen:

range of freshly prepared and aged solutions:
9.0 - 9.4 mg O2/L (control)
7.1 - 9.4 mg O2/L (solvent control)
4.7 - 9.3 mg O2/L (test concentrations)

Conductivity:

580 - 648 µS/cm between experimental day 0 and 19 (Dilution water)

Nominal and measured concentrations:

control, solvent control, 13.3, 24.0, 43.2, 77.8, 140 µg a.i./L (nominal)
for measured concentrations see Table 2 in section "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: covered with transparent glass plates
- Material, size, headspace, fill volume: 250 mL glass beakers (DIN 12332), individually labelled, filled with 100 mL of the test solution, corresponding to a fluid level of approximately 4 cm height;
- Renewal rate of test solution: 3 renewal intervals per week (on days 2, 5, 7, 9, 12, 14, 16 and 19)
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to Elendt, B.P. and Bias, W.R. (1990), Trace Nutrient Deficiency in Daphnia magna Cultured in Standard Medium for Toxicity Testing. Effects of the Optimisation of Culture Conditions on Life History Parameters of D. magna. Water Research 24 (9), 1157 1167
- Total organic carbon: < 2 mg/L
- Particulate matter: < 1 mg/L
- Metals: < 0.1 - 100 µg/L
- Pesticides: < 0.05 µg/L
- Chlorine: < 0.01 mg/L
- Alkalinity (as carbonate hardness): 3 German degrees [°dH]
- Ca/mg ratio: 6.25
- Culture medium different from test medium: no
- Intervals of water quality measurement: Samples were taken on experimental days 0, 2, 9, 12, 19 and 21

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

EFFECT PARAMETERS MEASURED: sublethal effects on parental animals and offspring, parental survival, time of brood release, offspring, parental body length and dry-body weight at study termination

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

13.3 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

24 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

28 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

24 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: parental age at first offspring emergence

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

43.2 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: parental age at first offspring emergence

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

13.3 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

24 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 140 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: final body length of surviving parental animals

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

140 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: final body length of surviving parental animals

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 140 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: final dry body weight of surviving parental animals

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 140 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: final dry body weight of surviving parental animals

Analytical results:

The accompanying chemical analysis of the test item in the freshly prepared test solutions at start of the chosen exposure intervals revealed recoveries between 99% and 115% (mean: 109%) of the corresponding nominal concentrations. The corresponding concentrations of the aged test solutions at the end of the exposure intervals ranged between 83% and 105% (mean: 94%) of nominal. As the measured concentrations were within the recommended range of 80 – 120 % of nominal, all reported results are based on nominal concentrations. No precipitants were observed in any of the test solutions. All measured values for the untreated control groups were found to be below the limit of quantification during analysis of the test samples (6.25 μg a.s. /L).

Table 1: Biological results (mean-values by study group):

	parental endpoints			reproductive endpoints
treatmentμg a.s./L (nominally)	body length (mm)	dry body mass (mg)	Immobility (%)	parent age at first offspring emergence (days)	total living offspring per parent animal (n)	total dead offspring per parent animal (n)
water control	4.1	0.568	0 %	9.78	93.4	0
solvent control (0.1 mL DMF/L)	4.1	0.500	0 %	9.58	92.6	0
13.3	4.1	0.484	10 %	9.98	90.7	0
24.0	4.0	0.468	20 %	10.38	78.0	0
43.2	4.0	0.580	30 %	10.58	59.0	1.2
77.8	4.1	0.555	20 %	7.78	3.2	42.8
140	4.1	0.583	30 %	9.78	0.0	74.1

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 May - 07 Jul 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Version / remarks:

draft; 1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, solvent control, 0.075, 0.15, 0.30, 0.60 and 1.2 mg a.i./L (nominal)
- Sampling method: Water samples were collected four and one days prior to the start. Water samples were collected from each treatment and control group at the beginning of the test, approximately weekly during the test and at test termination to measure concentrations of the test substance. Additional samples were collected on day 18 to confirm concentrations one day after gentle aeration was added to the test chambers. An additional set of samples were collected at termination. The samples were collected from mid-depth and placed in glass vials
- Sample storage conditions before analysis: direct analysis or stored refrigerated

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Initial stock solution: One stock solution per test concentrations, prepared seven times during the test. A primary stock solution, test item (60 mg a.i./L) was mixed with DMF, sonicated for 60 min and stirred for 30 min. Based on the primary stock solution, four secondary stock solutions (3.75, 7.5, 15 and 30 mg a.i./mL) were mixed with DMF and stirred for 30 min and stored refridgerated; one day after placing stock solution in the test system precipitation was observed and stock solutions were diluted to nominal concentrations of 0.75, 1.5, 3.0, 6.0 and 12 mg a.i./L and and placed back into the test system with an appropriate stock delivery flow rate.
Subsequent stock solutions: primary stock solution 12 mg/L solved in DMF, sonicated, and stirred; Four secondary stock solutions (0.75, 1.5, 3.0 and 6.0 mg a.i./mL) mixed with DMF and stirred. No further precipitation was observed.
- Controls: blank control, solvent control
- Chemical name of vehicle: DMF
- Concentration of vehicle in test medium: 0.01 mL/L
- Evidence of undissolved material: Undissolved material occurred five days prior to initiation of the test precipitate was observed in initial primary stock solutions after one day.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: mysid shrimp
- Source: Wildlife International was Reed Mariculture, Campbell, California; USA
- Age of parental stock (mean and range, SD): at least 14 d
- Feeding during test: yes four times daily
- Food type: live brine shrimp nauplii (Artemia sp.; INVE Aquaculture, Salt Lake City, Utah) (fed x 3) and brine shrimp enriched with a nutrient enrichment (fed x 1; A1 DHA Selco from INVE Thailand, Ltd., Amphoe Wachirabarami, Phichit, Thailand) and was also supplemented daily with Skeletonema costatum (cultured by Wildlife International)
- Amount: adjusted based on the density and age of the mysids throughout the study.
- Frequency: four times daily

ACCLIMATION
- Acclimation period: at least 14 d
- Acclimation conditions (same as test or not): same as test

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: the juvenile mysids (<24 h old) were collected and were impartially distributed one to three at a time into transfer containers until each container held 15 mysids

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

30 d

Test temperature:

25.6 - 26.2 °C (control)
25.5 - 26.4 °C (solvent control)
25.2 - 26.2 °C (test concentrations)

pH:

7.7 - 8.1 (control)
7.6 - 8.1 (solvent control)
7.6 - 8.0 (test concentrations)

Dissolved oxygen:

6.6 - 7.4 mg O2/L (control)
5.6 - 7.4 mg O2/L (solvent control)
5.0 - 7.4 mg O2/L (test concentration)

Salinity:

19 - 21 ‰ (measured only in the control)

Nominal and measured concentrations:

control, solvent control, 0.0075, 0.15, 0.30, 0.60 and 1.2 mg a.i./L (nominal)
control, solvent control, 0.0071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L (mean measured concentration)

Details on test conditions:

TEST SYSTEM
Test vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 2 L glass containers measuring 12 cm in diameter and 19 cm in height, with two nylon mesh covered holes on opposite sides of the container; compartments were placed in 9 L glass aquaria containing approximately 2.5 L of test solution. The depth of the water in a representative test chamber and test compartment was 6.2 and 5.8 cm, respectively; after sexual maturity (on day 13) reproductive pairs were placed in reproductive compartments, one pair per compartment, with up to five compartments in each replicate test chamber. An additional compartment was maintained in each test chamber, if necessary, to house any remaining males. The reproductive compartments were approximately 10 cm diameter glass petri dishes with sides of nylon mesh screen. The reproductive compartments were placed in 19 L glass aquaria filled with approximately 14.5 L of test solution, which contained a self-starting siphoning system to exchange test solution. The depth of the water in a representative test chamber and reproductive compartment was 17.0 and 16.6 cm, respectively.
- Aeration: Gentle aeration to each test chamber only on day 17 due to dissolved oxygen levels approaching 60% air saturation
- Type of flow-through (e.g. peristaltic or proportional diluter): Syringe pumps (Harvard Apparatus, Holliston, Massachusetts, USA) used to deliver stock solutions or solvent to the mixing chambers;
- Renewal rate of test solution (frequency/flow rate): juvenile test chambers: 18 volume additions of test water per day (stock solution 12.50 μL/minute and then mixed with dilution water delivered at a rate of 125 mL/minute); adult test chambers: at least 6 volume additions of test water per day (stock solution 25.0 μL/minute and then mixed with dilution water delivered at a rate of 250 mL/minute); flow rates varied by no more than ± 10% of the mean flow rate for the four replicates
- No. of organisms per vessel: 15 neonate mysids; after day 13 one pair per compartment
- No. of vessels per concentration (replicates): 4 with 1 compartment, after day 13 each replicate was subdivded in 5 compartments
- No. of vessels per control (replicates): 4 with 1 compartment; after day 13 each replicate was subdivded in 5 compartments
- No. of vessels per vehicle control (replicates): 4 with 1 compartment, after day 13 each replicate was subdivded in 5 compartments
- Biomass loading rate: 15 neonate mysid

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater collected at Indian River Inlet, Delaware, USA; collected seawater was pumped into holding tank and ozonated,before filtered through a sand filter to remove particles greater than approximately 25 μm, and pumped into a storage tank. filtered saltwater was then diluted to a salinity of approximately 20‰ with freshwater from a well on the Wildlife International site and was aerated with spray nozzles. Prior to use in the test, the 20‰ water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: < 2 mg C/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Salinity, dissolved oxygen was measured daily; pH and temperature was measured at test start and test end and approximately weekly during the test i9n case of temperature

OTHER TEST CONDITIONS
- Photoperiod: 14 h of light and 10 h of darkness with a 120 min transition period of low light
- Light intensity: wavelengths similar to natural sunlight (182 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): survival, reproduction and growth; the 7-, 14-, 21- and 28 day LC50 values were determined, when possible, based on the mortality of the G1 mysids.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
Range finding study
- Results used to determine the conditions for the definitive study: test concentrations were based on the results of an exploratory range-finding test

Reference substance (positive control):

no

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: G1 Mysid

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: G1 Mysid

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

> 1.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Observations on body length and weight:
Males: The mean total length of male mysids in the pooled controls and the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 8.17, 8.05, 8.17, 8.15, 8.09 and 8.33 mm, respectively. The mean dry weight of males in the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 0.79, 0.84, 0.91, 0.88 and 0.96 mg, respectively.
Femals: The mean total length of female mysids in the pooled controls and the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 8.27, 8.18, 8.54, 8.36, 8.37 and 8.19 mm, respectively. The mean dry weight of females in the pooled controls and the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 1.25, 0.99, 1.20, 1.16, 1.21 and 1.09 mg, respectively.

- Other biological observations:
G1 Juvenile Survival to Pairing: Surviving mysids in the control groups and in the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups appeared normal during the period from test initiation to pairing on Day 13.
G1 Adult Survival after Pairing: Surviving mysids in the treatment groups appeared normal during this period. There were a few observations of organisms that exhibited lethargy or erratic swimming behavior. However, these observations were infrequent and were not considered to be treatment related.

G1 Reproduction: The day of first brood release in this study was day 15. The percent of females producing young in the pooled controls and in the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 83.8, 100, 94.1, 58.8, 66.7 and 33.3%, respectively. The mean number of young produced per female in the pooled control groups and the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 6.2, 6.9, 8.3, 2.7, 4.1 and 1.6, respectively. The mean number of young produced per reproductive day in the pooled controls and in the 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.i./L treatment groups was 0.386, 0.431, 0.518, 0.157, 0.235 and 0.087, respectively.
G2 Survival (96 h): Surviving mysids in the negative and solvent control groups and in the treatment groups appeared normal with a few observations of lethargy during the 96 h evaluation period.
- Mortality of control: 1 out of 60 organisms
- Other: Analytical and Biological results are summarized in the tables 1-5 within section "Any other information on results incl. tables"

Results with reference substance (positive control):

No reference substance was tested.

Reported statistics and error estimates:

Test endpoints analyzed statistically for G1 mysid survival, G1 mysid reproduction (the number of live young produced per reproductive day, the number of young produced per surviving female and the percent of surviving females producing young), and G1 mysid growth (total body length and dry weight). Discrete-variable data were analyzed using Fisher’s Exact tests to identify treatment groups that showed a statistically significant difference from the pooled control (p ≤ 0.05). All continuous-variable data, were consistent with a monotonic concentration response and were analyzed using the Jonckheere-Terpstra trend test applied in a step-down procedure. All continuous-variable data were evaluated for normality using the Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (a = 0.01). All continuous-variable data passed the assumptions of normality and homogeneity of variance. Those treatment means that were significantly different from the pooled or solvent control means were identified using Dunnett’s test (p ≤ 0.05). All statistical tests were performed using a personal computer with SAS (3) software.

Since no differences were detected between the two control groups (p > 0.05) for any of the parameters, except mean male dry weight, the control data were pooled for comparison among the treatment groups. A difference was detected between the two control groups for the mean male dry weight parameter (p ≤ 0.05); therefore comparisons were made to the negative and solvent control group and reported based on comparisons to the solvent control group.

Table 1: Measured Concentrations of the test susbtance in test medium

Nominal Test Conc (mg a.i./L)	Rep.	Sample Number (149A-251-)	Sampling Time (Days)	Measured Conc (mg a.i./L)1,2	Percent of Nominal2	Mean Measured Conc (mg a.i./L)	Mean Measured Percent of Nominal
Negative	C	1	0	<LOQ	--	--	--
Control	D	8	6	<LOQ	--
	A	15	13	<LOQ	--
	B	22	18	<LOQ	--
	C	29	21	<LOQ	--
	D	36	30	<LOQ	--
	A	43	31	<LOQ	--
Solvent	C	2	0	<LOQ	--	--	--
Control	D	9	6	<LOQ	--
	A	16	13	<LOQ	--
	B	23	18	<LOQ	--
	C	30	21	<LOQ	--
	D	37	30	<LOQ	--
	A	44	31	<LOQ	--
0.075	C	3	0	0.0725	96.7	0.071 ± 0.008	95
	D	10	6	0.0805	107	CV = 10.9%
	A	17	13	0.0808	108
	B	24	18	0.0684	91.2
	C	31	21	0.0686	91.5
	D	38	30	0.0858	114
	A	45	31	0.0707	94.2
0.15	C	4	0	0.152	102	0.15 ± 0.008	100
	D	11	6	0.152	101	CV = 5.20%
	A	18	13	0.156	104
	B	25	18	0.150	100
	C	32	21	0.142	94.7
	D	39	30	0.166	110
	A	46	31	0.145	96.4
0.30	C	5	0	0.274	91.4	0.28 ± 0.028	93
	D	12	6	0.295	98.4	CV = 10.0%
	A	19	13	0.274	91.4
	B	26	18	0.282	93.9
	C	33	21	0.252	84.0
	D	40	30	0.319	106
	A	47	31	0.234	78.1
0.60	C	6	0	0.575	95.8	0.58 ± 0.054	97
	D	13	6	0.623	104	CV = 9.29%
	A	20	13	0.652	109
	B	27	18	0.565	94.1
	C	34	21	0.566	94.3
	D	41	30	0.577	96.2
	A	48	31	0.480	80.0
1.2	C	7	0	1.16	97.0	1.1 ± 0.080	92
	D	14	6	1.17	97.4	CV = 7.32%
	A	21	13	1.19	99.1
	B	28	18	1.01	84.0
	C	35	21	1.16	96.9
	D	42	30	1.12	93.5
	A	49	31	0.992	82.7

1 The limit of quantitation (LOQ) was 0.0250 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard

(0.0250 mg a.i./L) and the dilution factor of the matrix blank samples (1.00).

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

Table 2: Summary of Survival of G1 Saltwater Mysids Exposed to the test substance

	Juvenile Survival Initiation to Day 7			Juvenile Survival to Pairing on Day 13
Mean Measured Concentration (mg a.i./L)	Number Originally Exposed	Number Surviving	Percent Survival	Number Originally Exposed	Number Surviving	Percent Survival
Negative Control	60	60	100	60	59	98.3
Solvent Control	60	60	100	60	59	98.3
Pooled Control	120	120	100	120	118	98.3
0.071	60	59	98.3	60	57	95.0
0.15	60	54	90.0*1	60	51	85.0*1
0.28	60	59	98.3	60	57	95.0
0.58	60	56	93.3*1	60	55	91.7*1
1.1	60	56	93.3*1	60	53	88.3*

* Statistically significant decrease in survival in comparison to the pooled control using Fisher’s Exact test (p ≤ 0.05).

1 While the decrease in survival was statistically significant in comparison to the pooled control, it was not considered to be treatmentrelated since the difference was slight, there were no differences were detected in the adult survival data and the values were within the historical control range up to pairing.

Table 3: Summary of Survival of G1 Saltwater Mysids Exposed to the test substance

	Adult Survival Pairing to Day 21			Adult Survival Pairing to Test Termination on Day 30
Mean Measured Concentration (mg a.i./L)	Number Alive at Pairing 2	Number Surviving	Percent Survival	Number Alive at Pairing 2	Number Surviving	Percent Survival
Negative Control	48	46	95.8	48	42	87.5
Solvent Control	48	42	87.5	48	39	81.3
Pooled Control	96	88	91.7	96	81	84.4
0.071	42	40	95.2	42	36	85.7
0.15	35	35	100	35	34	97.1
0.28	46	40	87.0	46	36	78.3
0.58	47	40	85.1	47	36	76.6
1.1	50	43	86.0	50	37	74.0

1 There were no statistically significant decreases in survival in comparison to the control using Fisher’s Exact test (p > 0.05).

2 The number alive at pairing may be less than the number surviving to Day 14 due to the fact that extra females that cannot be used to form pairs and any immature mysids are discarded at the time of pairing on Day 14.

Table 4: Summary of Reproduction of G1 Saltwater Mysids Exposed to the test substance

Mean Measured Concentration (mg a.i./L)	Mean Number of Young Produced Per Reproductive Day ± SD 1	Percent of Surviving Females Producing Young 2	Mean Number of Young Per Surviving Female ± SD 1,2
Negative Control	0.331±0.084	79.0	5.4±1.26
Solvent Control	0.441±0.166	88.9	7.1±2.67
Pooled Control	0.386±0.135	83.8	6.2±2.13
0.071	0.431±0.140	100	6.9±2.25
0.15	0.518±0.100	94.1	8.3±1.62
0.28	0.157±0.062*	58.8 4	2.7±1.04*
0.58	0.235±0.072 3	66.7 3	4.1±1.69 3
1.1	0.087±0.131*	33.3**	1.6±2.31*

* Statistically significant decrease in reproduction and mean number of young per surviving female in comparison to the pooled control using Dunnett’s test (p ≤ 0.05).

** Statistically significant decrease in percent of surviving females producing young in comparison to the pooled control using Fisher’s Exact test (p ≤ 0.05).

1 There were no statistically significant trends detected in any of the growth endpoints using Jonckheere-Terpstra’s trend test (p > 0.05).

2 Calculated based on the total number of surviving females present at test termination.Females that died prior to test termination and the young that they produced were excluded from the calculation of the mean percent of females producing young and the mean number of young per female.

3 While the decreases were not statistically significant in comparison to the pooled control, a treatment-related effect could not be precluded since the decreases (40, 20 and 34 % reductions compared to the pooled control in the mean number of young per reproductive day, percent surviving females producing young and number of young per surviving female, respectively) were consistent among the three reproductive endpoints and dose-responsive.

4 While the decrease was not statistically significant in comparison to the pooled control, a treatment-related effect could not be precluded since the decrease (30% reduction compared to the pooled control) was consistent among the three reproductive endpoints and dose-responsive.

Table 5: Summary of Growth of G1 Saltwater Mysids Exposed to the test substance at termination day 30

Mean Measured Concentration (mg a.i./L)	Mean Total Length ± SD (mm)		Mean Dry Weight ± SD (mg)
	Males 1	Females 1	Males 1	Females 1
Negative Control	8.25±0.293	8.24±0.154	1.05±0.081	1.29±0.043
Solvent Control	8.09±0.045	8.30±0.136	0.93±0.033	1.22±0.117
Pooled Control	8.17±0.213	8.27±0.138	--2	1.25±0.090
0.071	8.05±0.199	8.18±0.248	0.79±0.096*3	0.99±0.149*3
0.15	8.17±0.104	8.54±0.078	0.84±0.047	1.20±0.024
0.28	8.15±0.159	8.36±0.055	0.91±0.073	1.16±0.111
0.58	8.09±0.056	8.37±0.160	0.88±0.057	1.21±0.119
1.1	8.33±0.158	8.19±0.213	0.96±0.032	1.09±0.120

* Statistically significant decrease in comparison to the solvent control using Dunnett’s test (p ≤ 0.05).

1 There were no statistically significant trends detected in any of the growth endpoints using Jonckheere-Terpstra’s trend test (p > 0.05).

2 Due to a statistically significant difference between the negative and solvent control groups, the data was compared to the negative and solvent control groups separately and reported to the solvent control group.

3 While a statistically significant decrease was detected using Dunnett’s test, it was not considered to be treatment-related since the difference was extremely slight, not dose-responsive, there was no corresponding decrease in length and there was no statistically significant trend detected using Jonckheere-Terpstra’s trend test.

Validity criteria fulfilled:

yes

Description of key information

Freshwater:

NOEC (21 d) = 13.3 µg a.s./L (Key, M-538451-01-1, Daphnia magna, OECD 211, nominal, for the most sensitive parameter: i.e. number of living offspring produced per parent animal)

EC10 (21 d) = 28.0 µg a.s./L (Key, M-538451-01-1, Daphnia magna, OECD 211, nominal, for the most sensitive parameter: i.e. number of living offspring produced per parent animal)

Marine water:

NOEC (30 d) 0.15 mg a.s./L (Key, M-508258-01-1, Americamysis bahia, OPPTS 850.1350, mean measured, most sensitive endpoint: G1 mysid reproduction)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

13.3 µg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.15 mg/L

Additional information

Two experimental studies are available investigating the long-term toxicity of the test item to aquatic invertebrates (M-538451-01-1 and M-508258-01-1). One study was conducted with the freshwater species Daphnia magna (OECD 211, GLP) and one study was conducted with marine species Americamysis bahia (OPPTS 850.1350, GLP). Due to a low solubility the test item was dissolved with dimethylformamide (0.1 mL/L) in order to prepare the test solutions.

Daphnia magna was exposed in a semi-static test system for a period of 21 d (renewal on days 2, 5, 7, 9, 12, 14, 16 and 19) to nominal concentrations of 13.3, 24.0, 43.2, 77.8 and 140 µg a.s./L. For verification of the actual test item concentrations during exposure, water-samples from start and end of three representative exposure-intervals were analysed. The measured amounts of the test item in the freshly prepared test solutions at the start of the chosen exposure intervals revealed recoveries between 99% and 115% (mean: 109%) of the corresponding nominal concentrations. The corresponding concentrations of the aged test solutions at the end at the end of the exposure intervals ranged between 83% and 105% (mean: 94%) of nominal. As the measured concentrations were within the recommended range of 80 – 120% of nominal the reported results are based on nominal concentrations of the test item. Following exposure, the total living offspring per parental animal, the parental age at first offspring emergence as well as the rate of parental survivors and their body-length and dry body mass were examined at the end of the study. The overall chronic NOEC after 21 d 13.3 µg a.s./L (nominal). This NOEC is based on total number of living offspring produced per parent animal and on immobilisation of parent animals. The corresponding LOEC (21 d) is 24.0 µg a.s./L (nominal). The EC10 (21 d) for the most sensitive endpoint, i.e. number of living offspring produced per parent animal, is 28.0 µg a.s./L (nominal). 

In the second study the first generation (G1) juveniles of Americamysis bahia were exposed in a flow-through system for a period of 30 d to nominal concentrations of 0.075, 0.15, 0.30, 0.60 and 1.2 mg a.s./L (corresponding to mean measured concentrations of 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.s./L, respectively). On day 14 of the test, after mysids attained sexual maturity, male and female adults were paired in each treatment and control group, with a maximum of five reproductive pairs per replicate. Reproduction of the paired mysids was monitored through termination on day 30. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning of the test, approximately weekly during the test and at test termination. Measured concentrations of the samples ranged from approximately 78.1 to 114% of nominal. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 0.071, 0.15, 0.28, 0.58 and 1.1 mg a.s./L, representing 95, 100, 93, 97 and 92% of nominal concentrations, respectively. Observations for G1 mysid mortality and signs of toxicity were conducted daily throughout the test. At test termination, the total body lengths and dry weights of all surviving first-generation mysids were measured. Observations of the effects of the test item on survival, reproduction and growth were used to determine the effect concentrations. At the end of exposure a NOEC (30 d) based on G1 mysid reproduction of 0.15 mg a.s./L was derived. The corresponding LOEC (30 d) was 0.28 mg a.s./L.