1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

31 July 2013 to 21 August 2013

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

Draft, 1996a

Qualifier:

according to guideline

Guideline:

other: EPA OSCPP 850.1000 (Special Considerations for Conducting Aquatic Laboratory Studies)

Version / remarks:

1996b

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the definitive exposure, duplicate samples of newly prepared test solutions (test days 0, 2, 9, 16 and 19) were removed from each treatment level and control prior to division into replicate test vessels. Additionally, aged test solutions were sampled and analyzed on test days 2, 5, 12, 19 and 21 for determination of the test substance concentration. Duplicate samples from aged test solutions were removed from a composite of all available replicate vessels. Of the duplicate samples taken at each time point, one sample was analyzed for the thest substance while the duplicate was stored frozen as archive backup sample.

Vehicle:

no

Details on test solutions:

At exposure initiation and weekly thereafter, a 10.7 mg/L primary stock solution was prepared by adding 0.0321 g of the test material to 3.0 L of dilution water. The test material and approximately 500 mL of dilution water were heated to approximately 30° C and ultrasonicated for approximately 30 min until oily spots formed on the solutions’ surface. The solution was then mixed overnight using a magnetic stir plate and stir bar. The resulting solution was observed to be clear and colorless with a slight oily film on the solutions’ surface. The 10.7 mg/L stock solution was siphoned, avoiding the solutions’ surface. The exposure solutions were mixed using a glass rod for one minute and were observed to be clear and colorless with no visible undissolved test substance.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas
- Stage and instar at study initiation: < 24 hours
- Method of breeding: The adult daphnids used to produce offspring for this test (1) did not contain ephippia, (2) produced offspring prior to being 12 days old, (3) produced an average of at least three offspring per female per day over the seven days prior to exposure initiation, (4) were not used in any portion of a previous test and (5) had less than 20% mortality during the 48 hours prior to exposure initiation.
- Source: Laboratory cultures maintained at the test facility

CULTURING CONDITIONS:
- Culture vessels: 1 L glass containing approximately 0.80 L of water
- Lighting location and type: Surface of the culture solution; regulated photoperiod of 16/8 (on/off) hours by fluorescent bulbs
- Light intensity: 60 to 77 footcandles (650 to 830 lux)
- Temp.: 20 ± 2 ºC (maintain by water bath)
- Feeding during culture: Daily; 200 µL of unicellular green algae Ankistrodesmus falcatus (4.0E+07 cells/mL) in addition to a 50 µL suspension of YCT (yeast, cereal leaves and flaked fish food). Daphnids were fed increasing amounts of food with increasing age.

- Feeding during test : Daily; 200 µL of unicellular green algae, Ankistrodesmus falcatus (4.0E+07 cells/mL), in addition to a 50 µL suspension of YCT (yeast, cereal leaves and flaked fish food). This quantity of algal suspension (200 mL/daphnid/day) is equivalent to approximately 0.20 mg carbon/daphnid/day.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

150 - 170 mg/L as CaCO3 (all test and control concentrations)

Test temperature:

- 19 - 21 ºC (all test and control concentrations, daily temperature measurement)
- 17 ºC (minimum temperature of the environmental chamber, continuous temperature monitoring)
- 21 ºC (maximum temperature of the environmental chamber, continuous temperature monitoring )

pH:

7.9 - 8.6 (all test and control concentrations)

Dissolved oxygen:

7.2 - 9.5 mg/L (all test and control concentrations)

Conductivity:

550 - 640 µS/cm (all test and control concentrations)

Nominal and measured concentrations:

- Nominal concentrations: control, 0.19, 0.38, 0.75, 1.5, 3.0 mg/L
- Measured concentrations: < LOQ, 0.18, 037, 0.73, 1.5, 2.9 mg/L, respectively. See Table 1 in "Any other information on materials and methods incl.tables "

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beaker with 80 mL of the test solution
- Test solution renewal: Test solution renewals were performed at exposure initiation and three times per week there afterward afterward. Freshly prepared test solution was added to a second set of clean beakers until each vessel contained 80 mL of solution. Daphnids were carefully transferred from the aged solution into the freshly prepared test solutions and the food solutions were then added to the newly prepared solutions.
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

DILUTION WATER PARAMETERS
- Source/preparation: Culture and test dilution water were prepared in 1900 L batches by fortifying well water according to the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. Fortified water was discarded if not used within 14 days of preparation.
- Total organic carbon: 1.8 mg/L (in July) and 0.64 mg/L (in August)
- Hardness: 160 mg/L as CaCO3
- Alkalinity: 90 - 94 mg/L as CaCO3
- Conductivity: 580 - 670 µS/cm
- pH: 8.1 - 8.3
- Dissolved oxygen: 7.4 - 8.7 mg/L
- None of pesticides, PCBs and toxic metals have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Culture water was the same source as the dillution water
- Intervals of the culture and test dilution water quality measurement: on each batch of fortified water prior to use.

OTHER TEST CONDITIONS
- Light source and location: Fluorescent bulbs, at the solutions' surface
- Photoperiod: a 16-hour light / 8-hour dark photoperiod was maintained with a 15-minute transition period by using electronic timers
- Light intensity: 2.3 - 12 µE·m-2·s-1

WATER QUEALITY MEASUREMENTS
- Dissolved oxygen, temperature and pH were measured in each test and control solution at the beginning (new solutions) and end (aged solutions) of each renewal period.
- The temperature of the environmental chamber was continuously monitored throughout the study using a VWR minimum/maximum thermometer.
- Total hardness, alkalinity and conductivity were measured and recorded in the freshly prepared solutions of the highest nominal test concentration (3.2 mg/L) and the control at exposure initiation and weekly thereafter in the freshly prepared solutions. For days 14 and 21, hardness, alkalinity and conductivity measurements were performed in the 1.6 mg/L nominal treatment level due to 100% immobilization by test day 8 in the 3.2 mg/L nominal treatment level. On day 21 (exposure termination), all measurements were taken from aged solutions.

EFFECT PARAMETERS MEASURED
- The number of immobilized adult daphnids and observations of abnormal behavior were recorded daily. Immobilization was defined as a daphnid not able to swim within 15 seconds after gentle agitation of the test vessel. Numbers of offspring were determined upon the first brood release in any vessel and daily thereafter throughout the remainder the test. Offspring were removed, counted and discarded at each observation interval. In addition, the number of immobilized offspring and the time to first brood release were recorded for each treatment level and the control. The second brood of offspring for each concentration and control was evaluated microscopically to determine the number of males present. Observations of physical characteristics of test solutions (e.g., precipitate, cloudy solution) were recorded whenever the test organisms were observed.
- At exposure termination (day 21), the total body length of each surviving adult daphnid was measured. Daphnids were measured (to the nearest 0.05 mm) from the apex of the head to the base of the carapace spine using an dissecting scope. Daphnids were dried at 100ºC for approximately 24 hours in a Precision Oven and the dry weight of each surviving adult daphnid was measured (to the nearest 0.01 mg).

TEST CONCENTRATIONS
- Range finding study: Based on existing data and in consultation with the Study Sponsor

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Total body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

Mortality / immobility

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

Mortality / immobility

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

95% C. L.: 0.11 - .74 mg/L

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Total body length

Details on results:

BIOLOGICAL RESULTS
After 21 days of exposure, survival in the dilution water control organisms averaged 100%. The mean number of offspring released per surviving female organism in the dilution water control during the 21-day test was 188 offspring per female. As demonstrated by the performance of the control organisms, the exposure system provided conditions which were appropriate for promoting acceptable survival and reproduction of the test species and met the minimum standard criteria established by OECD guidelines (i.e., ≥ 80% survival and ≥ 60 offspring per female). The dilution water control daphnids released their first brood of offspring on test day 8. Time of the first brood release was within normal performance expectation for this species. Therefore, all of the above parameters were well within the acceptable ranges of the study guidelines.

SURVIVAL
Survival of 100, 100, 100, 90, and 0% was observed among daphnids exposed to the 0.18, 0.37, 0.73, 1.5 and 2.9 mg/L treatment levels, respectively. Survival among control daphnids was 100%. Statistical analysis (Fisher's Exact Test with Bonferroni-Holm's Adjustment) determined a significant difference in survival among daphnids exposed to the 2.9 mg/L treatment level compared to the control (100%). Therefore, the NOEC was determined to be 1.5 mg/L and the LOEC was determined to be 2.9 mg/L. The EC10, EC20 and EC50 values for survival were determined to be 1.5, 1.6 and 2.0 mg/L, respectively, with 95% confidence limits of 0.96 to 1.6, 1.2 to 1.7 and 1.8 to 2.1 mg/L, respectively. Due to the statistically significant survival effect observed in the 2.9 mg/L treatment level, this treatment level was excluded from further statistical analysis.

RELEASE OF FIRST BROOD
First brood release occurred on day 8 for the control and 0.18 mg/L treatment level and day 9 for all other treatment levels tested, with the exception of the 3.0 mg/L treatment level, where no offspring were released.

REPRODUCTION
Organisms exposed to the 0.18, 0.37, 0.73, and 1.5 mg/L treatment levels released a mean cumulative offspring per female of 202, 168, 166, and 10 respectively. The control released a mean cumulative offspring per female of 188. The coefficient of variation for the control fecundity was 8.3%. Statistical analysis (Dunn’s Test with Bonferroni’s Adjustment) determined a significant reduction in cumulative offspring per female among daphnids exposed to the 1.5 mg/L treatment level compared to the control (188 offspring per female). Therefore, the NOEC was determined to be 0.73 mg/L and the LOEC was determined to be 1.5 mg/L. The EC10, EC20 and EC50 values for reproduction were determined to be 0.35, 0.80 and 1.1 mg/L, respectively, with 95% confidence limits of 0.11 to 0.74, 0.75 to 0.82 and 1.0 to 1.1 mg/L, respectively.

Immobilization in the 1.5 mg/L treatment level was considered to be toxicant related as there were statistically significant reproductive effects in this concentration and the next concentration higher had 0% survival. Since this daphnid was not classified as inadvertently immobilized, the statistical analysis for reproduction was performed with and without the offspring produced by this daphnid in accordance with the testing guideline (OECD 2012). The resulting endpoints (i.e., ECx values, NOEC and LOEC) were the same and therefore this data will be presented excluding the offspring from replicate E of the 1.5 mg/L treatment level.

OFFSPRING SEX RATIO
Additionally, all offspring from the second brood for each treatment and control were evaluated for gender microscopically (OECD 211 Annex 7, 2012) . All offspring were determined to be female and no further investigation was considered necessary.

GROWTH
Total body length at exposure termination among control daphnids averaged 4.77 mm per daphnid. Total body length among daphnids exposed to the 0.18, 0.37, 0.73 and 1.5 mg/L treatment levels averaged 4.79, 4.68, 4.61 and 4.32 mm, respectively. Statistical analysis (Dunn’s Test with Bonferroni’s Adjustment) determined a significant difference in total body length among daphnids exposed to the 0.73 and 1.5 mg/L treatment levels compared to the control. Therefore, the NOEC was determined to be 0.37 mg/L and the LOEC was determined to be 0.73 mg/L. Since no concentration resulted in ≥ 10% reduction, the EC10, EC20 and EC50 values for total body length were empirically estimated to be > 1.5 mg/L, the highest mean measured concentration tested.
Dry weight at exposure termination among control daphnids averaged 1.00 mg per daphnid. Dry weight among daphnids exposed to the 0.18, 0.37, 0.73 and 1.5 mg/L treatment levels averaged 0.67, 0.74, 0.74 and 0.61 mg per daphnid, respectively. Statistical analysis (Dunn’s Test with Bonferroni’s Adjustment) determined a significant difference in dry weight among daphnids exposed to the 1.5 mg/L treatment level compared to the control (0.70 mg per daphnid). Therefore, the NOEC for dry weight was determined to be 0.73 mg/L and the LOEC was determined to be 1.5 mg/L. The EC10 value for dry weight could not be determined since no upper 95% confidence limit could be calculated. According to the testing guideline, ECx values need to meet certain criteria (i.e., confidence intervals must not be overly wide) to be considered robust data and the EC10 data did not meet this criterion. Since no concentration resulted in ≥ 20% reduction, the EC20 and EC50 values for dry weight were empirically estimated to be > 1.5 mg/L, the highest mean measured concentration tested.

Reported statistics and error estimates:

See "statistical analysis" in 'Any other informatio on materials and methods incl. tables'.

Table 2. Sex ratio of offspring released per female daphnid (Daphnia magna) in the second brood during the 21-day static renewal exposure to the test substance.

Replicate	Gender Ratio of Live Second Brood Offspring Released per Replicate Exposure Vessel (% female)
	Control	0.18	0.37	0.73	1.5	2.9
A	100	100	100	100	100	NA^a
B	100	100	100	100	100	NA
C	100	100	100	100	100	NA
D	100	100	100	100	NA	NA
E	100	100	100	100	NA	NA
F	100	100	100	100	NA	NA
G	100	100	100	100	NA	NA
H	100	100	100	100	NA	NA
I	100	100	100	100	100	NA
J	100	100	100	100	NA	NA
Total # of Males	0	0	0	0	0	0
Total # of Females	401^b	406	451	469	13	0

^aNA = not applicable due to lack of offspring.

^bTwo offspring were inadvertently compromised and not able to be identified.

Table 3. Mean body length of daphnids (Daphnia magna) at the termination of the 21-day static renewal exposure to the test substance.

Mean Measured Concentration (mg/l)	Length (mm)
	Control	0.18	0.37	0.73	1.5	2.9
Replicate
A	4.55	3.10	4.60	4.60	4.35	IM
B	4.80	4.80	4.70	4.65	4.15	IM
C	4.70	4.80	4.55	4.55	4.10	IM
D	4.80	4.90	4.65	4.70	4.25	IM
E	4.80	4.75	4.65	4.60	IM	IM
F	4.75	4.85	4.75	4.75	4.45	IM
G	4.80	4.70	4.80	4.50	4.25	IM
H	4.75	4.75	4.65	4.60	4.40	IM
I	4.75	4.85	4.75	4.50	4.45	IM
J	4.95	4.75	4.70	4.60	4.45	IM
Mean	4.77	4.79	4.68	4.61^a	4.32^a	NA
SD	0.10	0.06	0.08	0.08	0.13	NA

^aSignificantly reduced compared to the control based on Dunn’s Test with Bonferroni’s Adjustment (p-value significant at < 0.05).

“IM” denotes the observation of an immobilized adult daphnid.

SD = Standard Deviation.

NA = Not Applicable.

Table 4. Mean dry weight of daphnids (Daphnia magna) at the termination of the 21-day static renewal exposure to the test substance. 

Mean Measured     Concentration (mg/L)	Weight (mg)
	Control	0.18	0.37	0.73	1.5	2.9
Replicate
A	0.68	0.47	0.68	0.70	0.68	IM
B	0.66	0.71	0.68	0.78	0.58	IM
C	0.65	0.71	0.77	0.63	0.44	IM
D	0.66	0.68	0.79	0.79	0.69	IM
E	0.59	0.79	0.72	0.61	IM	IM
F	0.85	0.68	0.76	0.76	0.48	IM
G	0.70	0.60	0.74	0.69	0.72	IM
H	0.67	0.64	0.69	0.70	0.56	IM
I	0.75	0.59	0.67	0.83	0.64	IM
J	0.77	0.65	0.85	0.87	0.68	IM
Mean	0.70	0.67	0.74	0.74	0.61^a	NA
SD	0.07	0.06	0.06	0.08	0.10	NA

^aSignificantly reduced compared to the control based on Dunn’s Test with Bonferroni’s Adjustment (p-value significant at < 0.05).

“IM” denotes the observation of an immobilized adult daphnid.

SD = Standard Deviation.

NA = Not Applicable.

Table 5. Mean measured concentrations tested, mean survival, reproduction, total body length and dry weight of parental daphnids (Daphnia magna) during the 21-day static renewal exposure to the test substance.

Mean Measured Concentration (mg/L)	Mean Percent Survival	Mean Number of Offspring Released (SD)	Mean Total Body Length in mm (SD)	Mean Dry Weight inmg(SD)
Control	100	188 (16)	4.77 (0.10)	0.70 (0.07)
0.18	100	182 (65)	4.63 (0.54)	0.65 (0.09)
0.37	100	168 (11)	4.68 (0.08)	0.74 (0.06)
0.73	100	166 (08)	4.61 (0.08)^*	0.74 (0.08)
1.5	90	10 (13)^*	4.32 (0.13)^*	0.61 (0.10)^*
2.9^a	0*	NA	NA	NA

* Significantly reduced compared to the control, based on Fisher's Exact Test with Bonferroni-Holm's

Adjustment (survival; p-value significant at < 0.05) or Dunn’s Test with Bonferroni’s Adjustment

(reproduction and growth; p-value significant at < 0.05).

^a Due to the survival effect observed, this treatment level was excluded from further statistical analysis.

SD = Standard Deviation.

NA = Not Applicable.

 

Table 6.The No-Observed-Effect Concentration (NOEC), Lowest-Observed-Effect Concentration (LOEC), and EC10, EC20 and EC50 values and corresponding 95% confidence limits established during the 21-day static renewal exposure to the test substance.

BiologicalParameter	Based on Mean Measured Concentrations (mg/L)
	NOEC	LOEC	EC10(95% CL^a)	EC20(95% CL)	EC50(95% CL)
21-Day Survival	1.5	2.9	1.5 (0.96 - 1.6)	1.6 (1.2 - 1.7)	2.0 (1.8 - 2.1)
21-Day Reproduction	0.73	1.5	0.35 (0.11 - 0.74)	0.80 (0.75 - 0.82)	1.1 (1.0 - 1.1)
21-Day Total BodyLength	0.37	0.73	> 1.5(NA^b)	> 1.5(NA)	> 1.5(NA)
21-Day Dry Weight	0.73	1.5	ND^c	> 1.5(NA)	> 1.5(NA)

^a CL = Confidence Limits.

^b NA = Not Applicable. EC value was empirically estimated, therefore, corresponding 95% confidence limits could not be determined.

^c ND = Not Determined. An upper 95% confidence limit could not be calculated failing to meet the estimation of ECx criteria and therefore an EC10 cannot be reported.

Validity criteria fulfilled:

yes

Conclusions:

Based on the test substance mean measured concentrations, the 21-day EC10s for survival and reproduction were determined to be 1.5 mg/L (with corresponding 95% confidence limites of 0.96 - 1.6 mg/L) and 0.35 mg/L (with corresponding 95% confidence limites of 0.11 - 0.74 mg/L), respectively. Based on growth (the total body length), the 21-day NOEC was determined to be 0.37 mg/L and the 21-day LOEC was determined to be 0.73 mg/L.

Executive summary:

This study was performed to assess the effect of the test substance on the survival, growth, reproductive output and offspring sex ratios of the water flea, Daphnia magna. The exposure was conducted under static renewal conditions following OECD Guideline for Testing of Chemicals 211, OCSPP Draft Guideline 850.1300 and OCSPP Guideline 850.1000. Ten replicate vessels, each containing one daphnid, were established for each treatment level and the control. Nominal concentrations for the treatment levels tested were 0.20, 0.41, 0.80, 1.6, and 3.2 mg/L (0.18, 0.37, 0.73, 1.5 and 2.9 mg/L as mean measured concentrations). Test vessels were maintained in a temperature-controlled environmental chamber set at 20 ± 1 °C with a 16 hour light and 8 hour dark photoperiod at a light intensity of 2.3 to 12 µE·m^-2·s^-1 (170 to 890 lux). The number of immobilized adult daphnids and observations of abnormal behavior in each test vessel were recorded daily. Numbers of offspring were determined upon the first brood release in any vessel and daily throughout the remainder the test. In addition, the number of immobilized offspring and the time to first brood release were recorded for each treatment level and the control. The female to male ratio in the second brood of offspring for each concentration and control was also assessed.

At exposure termination (day 21), the total body length of each surviving adult daphnid was measured and dry weights were determined. Results of this study were used to determine the NOEC (No-Observed-Effect Concentration) and LOEC (Lowest-Observed-Effect Concentration). The EC10, EC20 and EC50 values for survival, growth (total body length and dry weight) and reproduction were also determined where possible. The EC10, EC20 and EC50 values are defined as the concentration of the test substancen in dilution water which causes 10, 20 and 50% effect in the measured parameter, in the exposed test population after a fixed period of time. The results of this study are based on mean measured concentrations. Based on the test substance mean measured concentrations, the 21-day EC10s for survival and reproduction were determined to be 1.5 mg/L (with corresponding 95% confidence limites of  0.96 - 1.6 mg/L) and 0.35 mg/L (with corresponding 95% confidence limites of  0.11 - 0.74 mg/L), respectively. Based on growth (the total body length), the 21-day NOEC was determined to be 0.37 mg/L and the 21-day LOEC was determined to be 0.73 mg/L.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

31 Jul 1981 to 28 Aug 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method was performed in accordance with the test facility's Test Protocol for Mysid Shrimp: Acute and Life-Cycle Toxicity Test with Mysid Shrimp (Mysidopsis bahia)

GLP compliance:

no

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.114 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

The measured concentrations of the test substance in the seawater after different time intervals ranged from 49 to 122% of the nominal values and the mean measured concentrations were 82 - 101% of the nominal concentrations. The mortality was significantly greater in the two highest concentrations and 10 - 25% reduced in the other concentrations than in the control. On the basis of the reproduction results LOEC - and NOEC -values were 0.205 mg/L and 0.114 mg/L.

 

Table 1. The mortality of the shrimps based on the mean measured concentrations in the control and in the test substance exposed groups (%)

Day	Control		0.054		0.114		0.205		0.507		0.882
	A	B	A	B	A	B	A	B	A	B	A	B
7	0	0	0	0	20	0	10	20	30	40	80	90
14	0	10	0	10	30	0	10	20	50	80	100	60
21	0	10	0	10	30	20	10	20	70	90	100	70
28	0	0	10	10	30	20	10	20	70	90	100	80
Average	10		10		25		15		80*		90*

A and B = duplicates

* significantly greater than in the vehicle control (P ≤ 0.05)

 

Table 2. Reproduction results of shrimps based on the mean measured concentrations in the vehicle control and in the groups exposed to the test substance

Concentration (mg/L)	Number of offspring		Total offspring	Female with brood pouches	Offspring/female
	A	B
Vehicle control	10	19	29	9	3.2
0.054	16	11	27	8	3.4
0.114	21	9	30	9	3.3
0.205	0	5	5	4	1.2
0.507	0	0	0	20	0*
0.882	0	0	0	0	0*

A and B = duplicates

* significantly greater than in the vehicle control (P ≤ 0.05)

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 28-d NOEC of the substance is determinded to be 0.114 mg/L.

Executive summary:

The chronic effects of the test substance to mysid shrimp (Mysidopsis bahia) (< 48 hours old) was studied under a flow through conditions at the nominal concentrations of 0.062, 0.125, 0.25, 0.5 and 1 mg/L. The study was performed according with the test facility's Test Protocol for Mysid Shrimp: Acute and Life-Cycle Toxicity Test with Mysid Shrimp (Mysidopsis bahia). It was not compliant with GLP. The test included the vehicle control (acetone max 0.026 mg/L). There were 20 shrimps in each concentration level. The treated aquarias and the control were maintained at a temperature of 26 - 28°C, 7.8 - 7.9 pH, dissolved oxygen 4.2 - 6.3 mg/L (60 - 90 % of the saturation) and 27‰ salinity. Mortality and production of the offspring were recorded at 24 hours intervals. Salinity, temperature, pH and dissolved oxygen were measured daily. Water samples for chemical analyses of the test substance were taken alternatively from duplicates of each treatment level on days 0, 7, 14, 21 and 28.

The measured concentrations of the test substance in the seawater after different time intervals ranged from 49 to 122% of the nominal values and the mean measured concentrations were 82 - 101% of the nominal concentrations. The mortality was significantly greater in the two highest concentrations and 10 - 25% reduced in the other concentrations than in the control. On the basis of the reproduction results LOEC - and NOEC -values were 0.205 mg/L and 0.114 mg/L.

Description of key information

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

Freshwater; 21-d NOEC = 0.37 mg/L; Daphnia magna; growth (the total body length); OECD TG 211, Fournier, 2014

Marine water; 28-d NOEC= 0.114 mg/L; Mysidopsis bahia; mortality; no guideline followed; Hollister, 1981

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.37 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.114 mg/L

Additional information

There are two studies on freshwater invertebrates and one study on marine water invertebrates available for this endpoint. The 21-day study on Daphnia magna (Fournier 2014, OECD TG 211, GLP, Reliability 1) and 28-d study on mysid shrimp (Hollister 1981, no standard guidelines followed, not GLP, Reliability 2) were selected as key studies because they represent the worst-case effects (i.e. showed the lowest E(L)C50 value) for freshwater and marine water conditions, respectively.

In the freshwater key study, ten replicate vessels, each containing one daphnid, were used for each treatment level and the control. Nominal concentrations for the treatment levels tested were 0.20, 0.41, 0.80, 1.6, and 3.2 mg/L (0.18, 0.37, 0.73, 1.5 and 2.9 mg/L as mean measured concentrations, respectively). Test vessels were maintained at 20 ± 1 °C with 16-hour light and 8-hour dark photoperiod at a light intensity of 170 to 890 lux. The NOEC for growth was determined to be 0.37 mg/L based on mean measured concentrations. In the supporting study, the 21-day EC10 for the reproduction of Daphnia magna was determined to be 0.48 mg/L based on mean measured concentrations (LeBlanc & Mastone 1981, EPA guideline, GLP not specified, Reliability 2).

In the marine water key study, mysid shrimp (Mysidopsis bahia) were exposed to the test substance at nominal concentrations of 0.062, 0.125, 0.25, 0.5 and 1 mg/L (mean measured concentrations: 0.054, 0.114, 0.205, 0.507 and 0.882 mg/L, respectively). There were 20 shrimps in each concentration level. The test conditions were maintained at 26 - 28°C, pH 7.8 - 7.9, dissolved oxygen 4.2 - 6.3 mg O2/L and 27‰ salinity. The NOEC for reproduction was determined to be 0.114 mg/L based on mean measured concentrations.