Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

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:

disregarded due to major methodological deficiencies

Study period:

09 Nov - 07 Dec 2006

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Invalide study, validity criteria are not reached, the average offspring per female in the controls was only 0.77 per day indicating severe underperformance (performance criterion: >= 3/day). In the Joint review this study was considered invalid by PMRA and the EU RMS agreed in his evaluation for Europe.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Deviations:

yes

Remarks:

, validity criteria are not reached, the average offspring per female in the controls was only 0.77 per day indicating severe underperformance (performance criterion: >= 3/day).

Qualifier:

according to guideline

Guideline:

other: FIFRA Guideline Number 72-4

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples were taken from a point midway between the surface, bottom and sides of each vessel using a pipet.
- Exposure concentrations were analytically confirmed on days 0, 1, 7, 13, 21, 22 and 28 and were used to define measured exposure concentrations for this study. Samples of the aged (approx. 48 h old) and newly prepared diluter stock solutions (approx. 24 h old) were also analyzed at each sampling interval.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the test substance was prepared daily during the study by adding the test substance directly to artificial seawater. The resulting stock solution was mixed overnight and was observed to be clear and colourless with no visible signs of undissolved test substance following preparation.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid shrimp
- Source: Laboratory cultures at Springborn Smithers (SSL Lot No. 06A107); original source: Aquatic BioSystems Inc., Fort Collins Co, USA
- Age at study initiation (mean and range, SD): ≤ 24 h
- Feeding during test: yes
- Food type: live brine shrimp (Artemia salina) nauplii, twice daily
- Amount/frequency: During the period prior to pairing, at least one of these feedings was with brine shrimp nauplii enriched with Selco, a substance high in saturated fatty acids. Following the pairing phase, the mysids were fed brine shrimp nauplii enriched with Selco every other day.

CULTURE CONDITIONS
- Mysids were cultured in one of several 76-L glass aquaria with a closed-loop recirculating filtration system providing artificial filtered seawater to the aquaria. The seawater in the aquaria was characterized as having a salinity of 22 to 24‰ and a pH range of 8.1 to 8.3 during the 14-day period prior to test initiation. Dissolved oxygen percent saturation ranged from 98 to 102%, and specific conductance ranged from 32,000 to 35,000 µmhos/cm. The area in which the mysids were cultured received a regulated photoperiod of 16 hours of light and 8 hours of darkness.
- Type and amount of food: live brine shrimp (Artemia salina) nauplii; the first feeding was brine shrimp and the second feeding was brine shrimp enriched with Selco, a substance high in saturated fatty acids.
- Feeding frequency: ad libitum, twice daily

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

152 - 182 mg/L as CaCO3 (values for newly prepared batches of artificial seawater)

Test temperature:

24 - 27 °C

pH:

8.1 - 8.3

Dissolved oxygen:

5.1 - 7.3 mg/L

Salinity:

19 - 21 ‰

Nominal and measured concentrations:

Nominal: 0.19, 0.38, 0.75, 1.5, 3.0 mg a.i./L
Mean measured: 0.17, 0.31, 0.54, 1.1, 1.7 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Material, size, headspace, fill volume: size: 39 x 20 x 25 cm
- Renewal rate of test solution (frequency/flow rate): During each cycle of the diluter system, approximately 500 mL of exposure solution was delivered to each replicate test vessel. During the study, the diluter provided the exposure solutions to each test vessel at a rate of approximately 7.5 aquarium volume additions per day to provide a 90% test solution replacement rate of approximately 7.0 hours. The function of the diluter system (e.g., dilution water flow rates, stock solution consumption) was monitored daily and a visual check was performed twice each day. In this study, the flow-splitting accuracy was within 5% of the targeted delivery.
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.0027 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Artificial seawater was prepared by the addition of a commercially prepared salt formula (i.e., hw-MARINEMIX) to laboratory well water. Newly prepared batches of artificial seawater had a total alkalinity range of 152 to 182 mg/L as CaCO3, a specific conductance range of 20,000 to 27,000 µmhos/cm and a pH range of 8.0 to 8.2. The freshwater was mixed with the appropriate amount of salt in a 1700-L container to a salinity of 17 to 22‰. The artificial seawater was mixed and aerated vigorously for approximately 24 h, then allowed to aerate for an additional 24 h prior to use. The volume of dilution water which was prepared depended on the requirements of the diluter system.
- Total organic carbon: 0.37 mg/L for Nov 2006
- Toxic Metals: below levels of concern
- Pesticides: below levels of concern
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, dissolved oxygen, pH and salinity were measured on each batch of artificial seawater prior to use and daily in each test vessel.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/8 h dark
- Light intensity: 49 - 85 footcandles (530 - 920 lux) provided by Sylvania Oktron fluorescent bulbs

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After males and females had been paired (day 13), the number of dead males and females, the number of offspring produced by each individual female, and any abnormal appearance or behaviour was recorded daily. After test end, body length and dry weight was determined.

RANGE-FINDING STUDY
- 2 studies were performed:
- Test concentrations: (1) nominal 1.6, 3.1, 6.3, 13 and 25 mg a.i./L; (2) 6.3, 13, 25, 50 and 100 mg a.i./L
- Results used to determine the conditions for the definitive study: Since a NOEC level was not established, nominal concentrations were revised to 0.19, 0.38, 0.75, 1.5 and 3.0 mg a.i./L.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

other: MATC

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

- No. of offspring produced per day per female: validity criteria are not reached, the average offspring per female in the controls was only 0.77 per day indicating severe underperformance (performance criterion: >= 3/day).
- In general, the stability of stock solutions was questionable for a period of greater than 48 h, since degradation of the samples was observed. All stock solutions used during this study were < 48 h old.

Table: First generation (F0) survival, F0 reproduction, F0 average body length and F0 average dry body weight at the termination of the 28 day life-cycle test exposing mysids to AE 0001789 technical. (a) Significantly reduced compared to the control, based on Williams' Test.

Mean measured concentration (mg a.i./L)	Test Day 28 Survival (%, male-female combined)	Reproductive Success (Offspring/Female/Reproductive Day)	Average Total Body Length (mm)		Dry Body Weight (mg)
			Males	Females	Males	Females
Control	87	0.77	7.2	7.4	0.81	1.12
0.17	92	0.64	7.3	7.5	0.81	1.12
0.31	93	0.51 (a)	7.4	7.5	0.88	1.08
0.54	96	0.57 (a)	7.3	7.4	0.82	1.18
1.1	84	0.27 (a)	7.2	7.3 (a)	0.86	1.06
1.7	94	0.26 (a)	7.1	7.3 (a)	0.86	0.96 (a)

Validity criteria fulfilled:

no

Remarks:

validity criteria are not reached, the average offspring per female in the controls was only 0.77 per day indicating severe underperformance (performance criterion: >= 3/day).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Mar - 01 Apr 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

, draft

Qualifier:

according to guideline

Guideline:

other: FIFRA 72-4 (b)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: A sample from batch preparations for new solution analysis and a composite from all replicates at each test level for old solution analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method of mixing: Electric Mixer (for 100 mg a.i./L solution) and manual stirring with spatula (for all other levels including the controls).
- Duration of Mixing: Overnight (for 100 mg a.i./L solution based on solubility experiments) and approximately 1 minute (for all other levels including the controls)
- Controls: Yes
- Test concentration separation factor: 2

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: In-house culture originally obtained from Aquatic Biosystems, Fort Collins, CO on August 6, 2003
- Organism Age at Initiation: First instar < 24 hours old neonates
- Feeding during test: yes
- Food type: Green algae (Pseudokirchneriella subcapitata) and Tetrafin®
- Amount and Frequency: algae once daily at a rate of = 2 x 10s algal cells/L of test solution and a slurry of Tetrafin® on Monday, Wednesday and Friday at a rate of = 0.02 mg per mL of test solution.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

154 - 186 mg/L as CaCO3

Test temperature:

19.5 - 21.0 °C

pH:

8.0 - 8.7

Dissolved oxygen:

7.6 - 11.8 mg/L (84 to 130 % saturation)

Nominal and measured concentrations:

Nominal concentrations: Control, 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg a.i./L
Measured concentrations: Control (< 0.61), 3.13 (3.63), 6.25 (7.21), 12.5 (14.1), 25.0 (27.7), 50.0 (57.2) and 100 (111 .6) mg a.i./L (ppm)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1 liter beakers containing approximately 900 ml test solution (multiple organism beakers); 250 ml beakers containing approximately 200 ml test solution (single organisms beakers)
- Material, size, headspace, fill volume: 1 liter beakers containing approximately 900 ml test solution (multiple organism beakers) and 250 ml beakers containing approximately 200 ml test solution (single organisms beakers); all glass
- Renewal rate of test solution: on day 3, 10 and 17
- No. of organisms per vessel: 5 organisms (for sublethal and survival effects assessment) and 1 organism (for sublethal, survival, reproduction and growth effects).
- No. of vessels per concentration (replicates): 3 replicates (for sublethal and survival effects assessment) and 10 replicates (for sublethal, survival, reproduction and growth effects assessment).
- No. of vessels per control (replicates): 3 and 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: At experimental start, weekly thereafter including experimental finish.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 45 to 62 footcandles (480 to 670 lux)

EFFECT PARAMETERS MEASURED: Observations for sublethal effects and survival made daily, reproductive output (neonates counts) occurred at the time of first brood release and on Monday, Wednesday and Friday there after (to include the day of termination), growth determinations made at the end of the exposure.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

57.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

111.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Behavioral and Morphological Observations:

Test Concentration Used for Analysis:                  Mean Measured Concentrations

Observations Made:                                                    No effects noted for any surviving organisms

 

Immobilization:

Percent Immobilized:                                                                                                     0%to 16%

Passed Criteria for Normality and Homogeneity of Variance:                                         No

Statistical Analysis:                                                                                                        Non-parametric

Treatment Groups Significantly Different (P = 0.5) Controls:                                          None

No Observed Effect Concentration (NOEC):                                                                111.6 mg a.i./L

Lowest Observed Effect Concentration (LOEC):                                                       >111.6mga.i./L

Control Health:                                                                                                              Control organisms exceeded the criteria of <20% immobility (OECD 1998)

 

Time to First Brood:

Mean Time to First Brood Range:                                                                                  7.0 to 7.6 days (excluding the 1 1 1.6 mg a.i./L level in which brood development did not occur)

Passed Criteria for Normality and Homogeneity of Variance:                                          No

Statistical Analysis:                                                                                                        Non-parametric

Treatment Groups Significantly Different(P =0.5) Controls:                                             111.6 mg a.i./L

No Observed Effect Concentration (NOEC):                                                                 57.2 mg a.i./L

Lowest Observed Effect Concentration (LOEC):                                                           111.6 mg a.i./L

 

Neonates per Adult Reproduction Day:

Mean number of Young/Adult Reproductive day range:                                                  21.5 to 23.7 (excluding the 1 1 1.6 mg a.i./L level in which brood development did not occur)

Passed Criteria for Normality and Homogeneity of Variance:                                          No

Statistical Analysis:                                                                                                        Non-parametric

Treatment Groups Significantly Different(P =0.5) Controls:                                             111.6 mg a.i./L

No Observed Effect Concentration (NOEC):                                                                 57.2 mg a.i./L

Lowest Observed Effect Concentration (LOEC):                                                           111.6mg a.i./L

Reproductive Criteria:                                                                                                    Mean number of live offspring produced per parent control animal surviving at the end of the test exceeded the established criteria of = 60 (USEPA 1996)

 

Growth:

Day of Evaluation:                                                                                                        Adult Length and Dry Weight at Termination

 

Length

Mean Range:                                                                                                                  4.19 to 5. 21 mm

Passed Criteria for Normality and Homogeneity of Variance:                                           Yes

Statistical Analysis:                                                                                                          Parametric

Treatment Groups Significantly Different(P =0.5) Controls:                                              111.6 mg a.i./L

No Observed Effect Concentration (NOEC):                                                                  57.2 mg a.i./L

Lowest Observed Effect Concentration (LOEC)                                                             111.6mg a.i./L

 

Day of Evaluation:                                                                                                         Adult Length and Dry Weight at Termination

 

Dry Weight

Mean Range:                                                                                                                  1.074 to 1.692mg

Passed Criteria for Normality and Homogeneity of Variance:                                           Yes

Statistical Analysis:                                                                                                        Parametric

Treatment Groups Significantly Different(P =0.5) Controls:                                              6.25a, 111.6 mg a.i./L (" not considered biologically significant since the three higher test concentrations were not statistically significantly different from the control group)

No Observed Effect Concentration (NOEC):                                                                  57.2 mg a.i./L

Lowest Observed Effect Concentration (LOEC)                                                             111.6mg a.i./L

 

Observations:

No other sublethal effects were noted for surviving organisms.

Conclusions:     The 21-day exposure to AE 0001789 resulted in a NOEC of 57.2 mg a.i. /L and a LOEC of 111.6 mg a.i./L based on reproduction and growth.

 

 

Table 1: Measured Test Concentrations of AE 0001789 During the 21-day Exposure of the Daphnia magna

Nominal Concentration (mg a.i./L)	Measured concentrations (mg a.i./L)
	Day 0	Day 3	Day 10	Day 17	Day 21	Mean	Standard Deviation	Percent of Nominal
Control (new)	<0.61	<0.61	0.61	<0.61	0.61	NA	NA	NA
Control (old)	---	<0.61	0.61	0.61	---
3.13 (new)	3.57	3.49	3.69	3.90	3.68	3.63	0.14	116
3. 13 (old)	---	3.49	3.57	3.69	---
6.25 (new)	7.05	7.14	7.36	7.74	7.30	7.21	0.26	115
6.25 (old)	---	6.97	6.92	7.22	---
12.5 (new)	14.0	14.1	14.7	14.7	13.9	14.1	0.38	113
12.5 (old)	---	13.7	14.0	13.8	---
25.0 (new)	27.2	27.9	28.4	28.7	27.2	27.7	0.62	111
25.0 (old)	---	27.5	27.1	27.3	---
50.0(new)	53.9	56.8	58.8	58.2	57.5	57.2	1.53	114
50.0 (old)	---	57.9	58.1	56.6	---
100 (new)	107.4	108.9	117.1	116.2	113.7	111.6	4.55	112
100 (old)	---	103.8	113.4	112.0	---

Table 2: Validity criteria for OECD 211

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.	The mortality of the parent animals in the control did not exceed 20% at the end of the test.	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	Mean number of live offspring produced per parent control animal surviving at the end of the test exceeded the established criteria of = 60.	yes

Note: The same validity criterion (20%) can be used for accidental and inadvertent parental mortality for the controls as well as for each of the test concentrations.

Validity criteria fulfilled:

yes

Remarks:

See Table 2 in "Anyother information onresults incl. tables".

Description of key information

NOEC (21 d): 57.2 mg a.i./L (arith. mean measured, reproduction, Daphnia magna, OECD 211)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

57.2 mg/L

Additional information

The long-term effects of the test substance to freshwater invertebrates were evaluated in a 21d GLP-study on Daphnia magna according to the OECD guideline 211 (M-255076-01-1). The test design was semi-static with a weekly test substance renewal. Daphnia magna were exposed to nominal concentrations of 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg a.i./L (measured concentrations: 3.63,7.21 , 14.1, 27.7, 57.2 and 111.6 mg a.i./L) alongside a negative control. A NOEC (21d) of 57.2 mg a.i./L (mean measured) and a LOEC (21d) of 111.6 mg a.i./L (mean measured) for the parameters reproduction and growth were determined.