1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

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 Apr 2014 to 2 Oct 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:

1996 (draft)

Deviations:

yes

Remarks:

See Deviations in 'Any other information on results incl. tables'.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals: Prior to initiation of the exposure, duplicate water samples were collected from each treatment and control group. Duplicate samples were also collected from the stock solution approximately weekly prior to exposure and throughout the study to confirm the stock solution concentration. Duplicate water samples were collected from alternating replicate test chambers in each treatment and the control group at the beginning of the test, approximately weekly during the test and at G1 and G2 termination to measure concentrations of the test substance.
- Sample method and storage conditions before analysis: All of the samples were collected from mid-depth, placed in glass vials. One set of samples were processed immediately for analysis or stored under refrigerated conditions until analysis. The second set of samples was collected and stored refrigerated as back-up samples for possible future analysis. On Day 15 of the study, a pump failure occurred in the main saltwater delivery tank, which caused an interruption in filtered saltwater delivery to the test system. Upon discovery of this malfunction, duplicate samples from each treatment and control group were collected at that time and again on Day 16 to confirm that this interruption did not impact the concentrations in the test chambers. The exposure system was maintained statically, without saltwater flows, for no more than approximately eight hours. Due to stock solution delivery malfunctions that occurred on Day 25 and 31 in the 2.5 mg/L treatment group, Day 30 in the 1.3 mg/L treatment group and Day 30 in the 10 mg/L treatment group, additional duplicate samples were collected at the time these malfunctions were discovered and again later in the day to confirm recovery of exposure concentrations.
- Sample method and storage conditions before analysis: All of the samples were collected from mid-depth, pla ced in glass vials. One set of samples were processed immediately for analysis or stored under refri gerated conditions until analysis. The second set of samples was collected and stored refrigerated as back-up samples for possible future analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A primary stock solution was prepared at a nominal concentration of 800 mg/L and proportionally delivered to each of the concentrations tested. The stock was prepared nine times during the test. The filtered saltwater (dilution water) used to prepare the stock solutions was first adjusted to a pH of 9.2 to 9.3 with a 0.1M NaOH solution prior in order to aid in solubilizing the test substance. The primary stock solution was then prepared by weighing and adding a calculated amount of test substance into one or two 30-L sheer pump holding tanks that contained a pre-measured volume of pH-adjusted dilution water and mixed by the forced vortex of the sheer pump for approximately four to six hours. The primary stock solution was stored ambient during mixing. After mixing, the solution was drained from the sheer pump and added to a 38-L glass aquarium. Once put into the exposure system, the stock solution was stirred gently and continuously with a top-down electric mixer. Newly prepared stock was placed on the exposure system every two to five days during the study.
Prior to pairing of the mysids, the appropriate amount of the 800 mg/L stock was injected into the diluter mixing chambers assigned to treatment groups at target rates of 0.0388, 0.0788, 0.16, 0.31, 0.63 and 1.25 mL/minute where they were mixed with dilution water delivered at target rates of 100, 100, 100, 100, 99 and 99 mL/minute to achieve the desired test concentrations. The negative control received dilution water only at a target rate of 100 mL/min. Following pairing, stock solution was injected into the diluter mixing chambers at target rates of 0.0782, 0.159, 0.33, 0.63, 1.25 and 2.53 mL/minute where they were mixed with dilution water delivered at target rates of 202, 202, 202, 201, 201 and 199 mL/minute to achieve the desired test concentrations. The negative control received dilution water only at a target rate of 202 mL/min.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid shrimp
- Age at study initiation: Juveniles (<24 hours old)
- Method of breeding: Adult mysids in the cultures were held in the laboratory for at least 14 days before neonates were collected for testing. Adult mysids held in the culture were evaluated prior to exposure initiation and used to collect young for the test were observed to be in excellent population health (e.g. high survival, high reproduction, etc.). The culture was maintained in a flow-through saltwater system using water from the same source as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 25.1 to 26.1ºC. The pH of the water ranged from 8.0 to 8.4. Dissolved oxygen concentrations were ≥6.5 mg/L (≥88% of saturation). Salinity of the filtered saltwater ranged from 20 to 21 parts per thousand (‰).
- Source: In-house culture
Feeding of culture: Daily with live brine shrimp nauplii (Artemia sp.). The brine shrimp were enriched daily with a nutrient enrichment.

FEEDING DURING TEST
- Food type: Live brine shrimp nauplii 24 to 48 hours old (Artemia sp.)
- Frequency: Up to four times daily.
- Amount: The amount of live brine shrimp nauplii (Artemia sp.) fed to the mysids was adjusted based on the density and age of the mysids throughout the study. The mysids in the test were fed the enriched brine shrimp for one of the daily feedings. Mysid food was also supplemented daily with Skeletonema costatum.
- Cleaning: Excess food and waste were siphoned out daily during observations.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

35 d

Remarks on exposure duration:

31 Days for G1 mysids; 96-hours for G2 mysids

Test temperature:

24.7 - 25.9 °C

pH:

7.8 - 8.1

Dissolved oxygen:

6.3 - 7.4 mg O2/L

Salinity:

19 - 21 ‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (blank control), 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg/L.
- Mean measured concentrations: < LOQ, 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L.
See Table 1 and Table 2 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel prior to pairing: 2-L glass containers measuring 12 cm in diameter and 19 cm in height, with two nylon mesh (220 μm mesh size) covered holes on opposite sides of the container. The compartments were placed in 9-L glass aquaria containing approximately 2 to 3 L of test solution. The depth of the water in a representative test chamber and test compartment was 5.9 and 5.1 cm, respectively.
- Test vessel after pairing: After mysids attained sexual maturity and were paired 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 (220 μm mesh size). 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 18.3 and 17.1 cm, respectively. An additional compartment (constructed exactly as the reproductive compartments) was maintained in each test chamber to house any G2 mysids for a 96-hour evaluation period, when available.
- Aeration: No
- Type of flow-through: Proportional diluter
- Renewal rate of test solution: The flow of dilution water into each mixing chamber was adjusted to provide each juvenile test chamber with at least 14 volume additions of test water per day and adult test chambers with at least 5 volume additions of test water per day. After mixing, the flow from each mixing chamber was split to deliver test water to each replicate test chamber.
- No. of organisms per vessel: 15 until day 13. On Day 13 of the test, after G1 mysids attained sexual maturity, male and female adults were paired in each treatment and the control group, with a maximum of five reproductive pairs per replicate. During the reproductive phase, when available, groups of G2 offspring were collected from the G1 replicate test chambers and were maintained in a separate test compartment within each G1 replicate test chamber. Each G2 test compartment contained up to 10 G2 offspring for a total of up to 15 G2 offspring per replicate and 40 G2 offspring per treatment and the control group, when available.
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM
- Source/preparation of dilution water: The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware. The freshly-collected seawater was pumped into a 5000-gallon holding tank and ozonated, before filtered through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800-L storage tank. The filtered saltwater was then diluted to a salinity of approximately 20‰ with freshwater from a well on the test facility site and was aerated with spray nozzles. Prior to use, the 20‰ water was filtered to 0.45 μm to remove fine particles and was then passed through an ultraviolet (UV) sterilizer.

WATER PARAMETERS
- Temperature: Recorded continuously in one negative control test chamber and recorded in each test chamber at the beginning of the test and end of the G1 mysid exposure period, and approximately weekly during the test.
- Dissolved oxygen (D.O.): Prior to pairing, dissolved oxygen was measured in one replicate test chamber of each treatment and the control group at the beginning of the test and approximately weekly during the exposure period, with measurements rotating among the replicates in each group at each measurement interval. After mysids attained sexual maturity and were paired on Day 13, dissolved oxygen was measured daily until the end of the test in one replicate test chamber of each treatment and the control group, with measurements rotating among the replicates in each group at each measurement interval.
- pH: Measurements of pH were made in one replicate test chamber of each treatment and the control group at the beginning and end of the G1 mysid exposure period, and approximately weekly during the test, with measurements rotating among the replicates in each group at each measurement interval.
- Salinity: Salinity was measured daily in one replicate of the negative control, with measurements rotating among the replicates in the group at each measurement interval.

OTHER TEST CONDITIONS
- Adjustment of pH: The filtered saltwater (dilution water) used to prepare the stock solutions was first adjusted to a pH of 9.2 to 9.3 with a 0.1M NaOH solution prior in order to aid in solubilizing the test substance.
- Photoperiod: 14 hours of light and 10 hours of darkness daily. A 120-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.
- Light intensity: Light intensity at exposure initiation was 146 lux at the surface of the water of one representative test chamber in the negative control group replicate A.

EFFECT PARAMETERS MEASURED:
Observations of the survival and behavior of each first-generation mysid were made daily throughout the test. The criteria for death included lack of movement, absence of respiratory movements, and lack of reaction to gentle prodding. At pairing on Day 13, the sex and maturity of each mysid was determined by microscopic examination, and when possible, five male/female pairs were assigned to reproductive compartments in each replicate test chamber, with one pair per compartment. Any immature mysids or extra females were discarded at this time. Any sexually mature males remaining after pairing were maintained in a separate compartment within the respective replicate test chamber. If a male in a reproductive compartment died, it was replaced with a male from the pool of males maintained in the same replicate, if available.
Following pairing, second-generation mysids produced in each compartment were counted, recorded and removed daily. Second-generation mysids were also observed for abnormal development and abnormal behavior. The G1 mysids were terminated on Day 31, which was at least seven days past the median time of first brood release for the negative control (Day 22). At test termination, the sex of each surviving first-generation mysid was confirmed and the total length of each mysid was measured to 0.01 mm using calipers. The mysids then were placed in a drying oven, set at approximately 60°C. The mysids were dried for approximately 69 hours. After drying, the mysids were then measured for dry weight to 0.01 mg on an analytical balance.
When available, one group (n≤10 per compartment) in each replicate of each control and test concentration of G2 mysids produced during the reproductive phase were maintained under their respective test conditions and observed for mortality, signs of toxicity, appearance, and behavior for approximately 96 hours post release. The G2 mysids were collected from multiple G1 pairs, preferentially within each replicate, to obtain up to 15 offspring per replicate and up to 40 offspring per treatment and control groups, when available. However, when necessary, offspring produced on the same day were collected and pooled from different replicates per concentration to obtain the recommended number of offspring. The G2 offspring were collected up until Day 28 of the test.

Reference substance (positive control):

no

Key result

Duration:

31 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

31 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

juveniles (day 0-13) and adults (day 14-31)

Key result

Duration:

31 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

length and weight

Details on results:

An overview of the results is provided in Tables 3 - 5 on 'Any other information on results incl. tables'
- G1 Juvenile Survival to Pairing (Days 0 - 13): In general, surviving mysids in the control and treatm ent groups appeared normal during the period from exposure initiation to pairing on Day 13. There wa s one observation of an organism that exhibited erratic swimming in the 5.3 mg/L treatment groups. T his observation was not concentration-responsive, therefore, not considered to be treatment-related. After 7 and 13 days of exposure, survival of mysids in the negative control was 100 and 98.3%, respectively. After 7 days of exposure, survival in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L groups was 100, 98.3, 96.7, 98.3, 95.0 and 93.3%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any treatment groups when compared to the negative control (p > 0.05). After 13 days of exposure, survival in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L groups was 98.3, 98.3, 95.0, 91.7, 91.7 and 93.3%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any treatment groups when compared to the negative control (p > 0.05). The LOEC for juvenile survival (Days 0 –13) was >10 mg/L, and the NOEC was 10 mg/L. There was less than 50% mortality in any of the treatment groups tested through Day 13 of the study. Therefore the 7- and 13-day LC50 values were empirically estimated to be >10 mg/L, the highest concentration tested.
- G2 Adult Survival After Pairing (Days 14 - 31): In general, surviving mysids in the negative control and the treatment groups appeared normal during this period. There were a few observations of o rganisms that exhibited lethargy in the negative control, 0.33, 1.2, 2.5, 5.3 and 10 mg/L treatment groups. However, these observations were infrequent and were not considered to be treatment related. After 21 and 31 days of exposure, survival of mysids in the negative control was 83.7 and 73.5%, respectively.
After 21 days of exposure, survival in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L groups was 94.2, 90.7, 93.6, 95.3, 96.0 and 97.6%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any treatment groups when compared to the negative control (p > 0.05). After 31 days of exposure, survival in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L groups was 78.8, 76.7, 78.7, 81.4, 80.0 and 78.6%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any treatment groups when compared to the negative control (p > 0.05). The LOEC for adult survival (Days 14 –31) was >10 mg/L, and the NOEC was 10 mg/L. There was less than 50% mortality in any of the treatment groups tested through Day 31 of the study. Therefore the 21- and 31-day LC50 values were empirically estimated to be >10 mg/L, the highest concentration tested.
- G1 Reproduction: The day of first brood release in this study was Day 15. The percent of surviving females producing young, mean number of young produced per surviving female and mean number of young produced per reproductive day in the negative control was 100, 13.3 and 0.774, respecti vely. The percent of surviving females producing young in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L treatment groups was 100, 100, 100, 93.8, 93.8 and 88.2%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in percent of surviving females reproducing young in any of the treatment groups in comparison to the negative control (p >0.05). The mean number of young produced per surviving female in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L treatment groups was 14.5, 15.9, 12.0, 14.0, 12.7 and 11.5, respectively. Dunnett’s test indicated there were no statistically significant decreases in the mean number of young produced per surviving female in any of the treatment groups when compared to the negative control (p > 0.05). Jonckheere-Terpstra’s trend test indicated there was no statistically significant decreased trend detected in the data in any of the treatment groups (p > 0.05). The mean number of young produced per reproductive day in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L treatment groups was 0.890, 0.852, 0.602, 0.817, 0.761 and 0.680, respectively. Dunnett’s test indicated there were no statistically significant decreases in the mean number of young produced per reproductive day in any of the treatment groups when compared to the negative control (p > 0.05). Jonckheere-Terpstra’s trend test indicated there was no statistically significant decreased trend detected in the data in any of the treatment groups (p > 0.05). Consequently, the LOEC and NOEC for reproduction was >10 and 10 mg/L, respectively.
- G1 Growth: The mean total length and dry weight of male mysids in the negative control group was
7.60 mm and 1.15 mg, respectively. The mean total length of male mysids in the 0.33, 0.68, 1.2, 2.5,
5.3 and 10 mg/L treatment groups was 7.52, 7.58, 7.54, 7.74, 7.77 and 7.73 mm, respectively. The mean dry weight of males in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L treatment groups was 1.02, 1.20, 1.10, 1.03, 1.15 and 1.15 mg, respectively. Dunnett’s test indicated there were no statistically significant decreases in mean male total length or dry weight in any of the treatment groups when compared to the negative control (p > 0.05). Jonckheere-Terpstra’s trend test indicated there was no statistically significant decreased trend detected in the data in any of the treatment groups (p > 0.05).
The mean total length and dry weight of female mysids in the negative control group was 7.63 mm and 1.44 mg, respectively. The mean total length of female mysids in the 0.33, 0.68, 1.2, 2.5, 5.3
and 10 mg/L treatment groups was 7.56, 7.66, 7.59, 7.86, 7.71 and 7.59 mm, respectively. The mean
dry weight of females in the 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L treatment groups was 1.42, 1.38,
1.38, 1.46, 1.46 and 1.34 mg, respectively. Dunnett’s test indicated there were no statistically significant decreases in mean female total length or dry weight in any of the treatment groups when compared to the negative control (p > 0.05). Jonckheere-Terpstra’s trend test indicated there was no statistically significant decreased trend detected in the data in any of the treatment groups (p > 0.05). Consequently, the LOEC and NOEC for growth (lengths and weights) was >10 and 10 mg/L, respectively.
- G2 Survival (96-Hours): Biological observations of sublethal effects and survival of second-generat ion (G2) juvenile mysids from the day they were collected until approximately 96-hours after release from the brood pouch. Surviving mysids in the negative control group and in the treatment groups ap peared normal with a few observations of lethargy in the negative control, 0.68 and 1.2 mg/L treatment groups and lethargy and erratic swimming in the 5.3 and 10 mg/L treatment groups during the 96- hour evaluation period. These observations were infrequent and non-dose responsive, therefore these were not considered treatment-related. The data set was too small to establish any meaningful statisti cal relationships in the data and statistical analysis was not conducted.

Reported statistics and error estimates:

The continuous-variable data for the reproductive and growth endpoints were examined to determine whether the concentration-response was fundamentally monotonic (trending in one direction, e.g., response not trending up and then down as concentration increases) or non-monotonic. Monotonicity of the dose-response for each endpoint was determined by converting the variable responses to rank values, and then performing a regression of the response ranks versus concentration using a quadratic polynomial as the model. The concentration-response relationship for all of the data was considered to be monotonic, since the quadratic term was either not significant (p > 0.05) or both the linear and quadratic terms were significant (p ≤ 0.05).

Discrete-variable data were analyzed using Chi-square and Fisher’s Exact tests to identify treatment groups that showed a statistically significant difference from the negative 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 either Levene’s or Bartlett’s test (a = 0.01). The data for all of the continuous-variable data passed the assumptions of normality and homogeneity of variance. Those treatment means that were significantly different from the negative control means were identified using Dunnett’s test (p ≤ 0.05).

Table 3. Summary of Survival of Saltwater Mysids

 

Survival of Saltwater Mysids (a)
	Juvenile Survival Initiation to Day 7			Juvenile Survival to Pairing on Day 13
Mean Measured Concentration (mg/L)	Number Originally Exposed	Number Surviving	Percent Survival	Number Originally Exposed	Number Surviving	Percent Survival
Negative Control	60	60	100	60	59	98.3
0.33	60	60	100	60	59	98.3
0.68	60	59	98.3	60	59	98.3
1.2	60	58	96.7	60	57	95.0
2.5	60	59	98.3	60	55	91.7
5.3	60	57	95.0	60	55	91.7
10	60	56	93.3	60	56	93.3
(a) There were no statistically significant decreases in any of the treatment groups when compared to the negative control (p > 0.05).

Table 3. Summary of Survival of Saltwater Mysids - continued

Survival of Saltwater Mysids (a)
	Adult Survival Day 14 to Day 21			Adult Survival Day 14 to Test Termination on Day 31
Mean Measured Concentration (mg/L)	Number Alive at Pairing ^2	Number Surviving	Percent Survival	Number Alive at Pairing (b)	Number Surviving	Percent Survival
Negative Control	49	41	83.7	49	36	73.5
0.33	52	49	94.2	52	41	78.8
0.68	43	39	90.7	43	33	76.7
1.2	47	44	93.6	47	37	78.7
2.5	43	41	95.3	43	35	81.4
5.3	50	48	96.0	50	40	80.0
10	42	41	97.6	42	33	78.6
(a) There were no statistically significant decreases in any of the treatment groups when compared to the negative control (p > 0.05). (b) The number alive at pairing may be less than the number surviving to Day 13 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 13.

Table 4. Summary of Reproduction of Saltwater Mysids

Triasulfuron Mean Measured Concentration (mg/L)	Mean Number of Young Produced Per Reproductive Day ± SD ^1	Percent of Surviving Females Producing Young (a,b)	Mean Number of Young Per Surviving Female ± SD (a,b)
Negative Control	0.774 ± 0.127	100	13.3 ± 2.78
0.33	0.890 ± 0.138	100	14.5 ± 2.72
0.68	0.852 ± 0.252	100	15.9 ± 5.75
1.2	0.602 ± 0.058	100	12.0 ± 3.54
2.5	0.817 ± 0.272	93.8	14.0 ± 5.33
5.3	0.761 ± 0.183	93.8	12.7 ± 2.80
10	0.680 ± 0.090	88.2	11.5 ± 1.41
(a) There were no statistically significant decreases in any of the treatment groups when compared to the negative control (p > 0.05). (b) 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.

Table 5. Summary of Growth of Saltwater Mysids

Growth Parameters at Termination on Day 31 (a)
Triasulfuron Mean Measured Concentration (mg/L)	Mean Total Length ± SD (mm)		Mean Dry Weight ± SD (mg)
	Males	Females	Males	Females
Negative Control	7.60 ± 0.118	7.63 ± 0.094	1.15 ± 0.081	1.44 ± 0.111
0.33	7.52 ± 0.172	7.56 ± 0.279	1.02 ± 0.041	1.42 ± 0.148
0.68	7.58 ± 0.183	7.66 ± 0.108	1.20 ± 0.102	1.38 ± 0.044
1.2	7.54 ± 0.151	7.59 ± 0.204	1.10 ± 0.090	1.38 ± 0.210
2.5	7.74 ± 0.115	7.86 ± 0.126	1.03 ± 0.042	1.46 ± 0.064
5.3	7.77 ± 0.264	7.71 ± 0.182	1.15 ± 0.156	1.46 ± 0.150
10	7.73 ± 0.149	7.59 ± 0.155	1.15 ± 0.075	1.34 ± 0.130
(a) There were no statistically significant decreases in any of the treatment groups when compared to the negative control (p > 0.05).

Deviations

1. The protocol was amended from stating “groups of offspring will be counted and retained) to “groups of live and healthy offspring will be counted and retained” to clarify the protocol to current procedures.
2. During the reproductive phase of the study, there several instances when compartments in replicates were initiated with G2 offspring after 10 offspring had been initiated previously in the same replicate. The protocol states that groups of offspring (10 per replicate, if available) will be counted and retained in a common compartment for each replicate at the same test concentration as the G1 chambers where they originated. While the number of offspring initiated exceeded the required amount stated in the protocol per replicate, no more than 10 offspring were ever initiated in a compartment at a time. Additionally, each replicate had offspring that in which G2 offspring had been initiated. Therefore, this deviation from the protocol had no adverse impact upon the results or interpretation of the study.
3. G2 compartments were initiated past Day 24 of the study. The protocol states that all G2 groups will be collected by Day 24 of the test. While this was more data than what was required under the protocol, these data were valuable and helped in the evaluation of the G2 mysids. Therefore, this deviation from the protocol had no adverse impact upon the results or interpretation of the study.
4. On Day 25 and Day 31 in the 2.5 mg/L treatment group, Day 30 in the 1.3 mg/L treatment group and Day 30 in the 10 mg/L treatment group, a stock solution delivery malfunction occurred. The protocol states that mysids will be continuously exposed beginning at the juvenile stage through termination. Upon the discovery of these malfunctions, analytical samples were collected and analyzed. Based on the recoveries it was determined that delivery was interrupted briefly; therefore, time-weighted averages were calculated for the 1.3, 2.5 and 10 mg/L treatment groups. While there was an interruption in toxicant delivery, the interruption was minimal when compared to the length of the test. Therefore, it is our assertion that this deviation from the protocol had no adverse impact upon the results or interpretation of the study.

Validity criteria

The study is valid because the following criteria were met:

• ≤30% of the first-generation control mysids died during the course of the test


• ≥75% of the first-generation females in the control produced young


• The average number of young produced by the first-generation females in the control was ≥ three young per first-generation female

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'.

Conclusions:

In a life-cycle toxicity test with saltwater mysid, performed in accordance with EPA 850.1350 (draft), the 31-day NOEC for mortality, reproduction and growth was determined to be 10 mg/L, based on mean measured concentrations.

Executive summary:

The impact of the test item on the life-cycle of Americamysis bahia was studied in a flow-through system for 35 days (31 Days for G1 mysids; 96-hours for G2 mysids). The study was conducted in accordance with EPA 850.1350 (draft) and in compliance with  GLP criteria. The test organisms (< 24-hours old) were exposed to the test item in marine water at 24.7 - 25.9 ˚C, pH 7.8 – 8.1 and dissolved oxygen 6.3 – 7.4 mg O2/L with a salinity of 19-21 ‰ and a photoperiod of 14 hours daily (146 Lux at the start). The organisms (initially 15 shrimp/vessel; 4 vessels/concentration or control) were exposed to nominal concentrations of 0 (blank control), 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg/L of the test item. The concentrations of the test item were analysed using HPLC/UV-detection. The mean measured concentrations were 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L, respectively. Observations of the survival and behavior of each first-generation mysid were made daily throughout the test. Following pairing, second-generation mysids produced in each compartment were counted, recorded and removed daily. Second-generation mysids were also observed for abnormal development and abnormal behavior.

There were no statistically significant decreases in G1 mysid adult survival, reproduction or growth in any of the treatment groups compared to the negative control. There was less than 50% mortality in any of the treatment groups tested through termination of the G1 mysid exposure phase. Therefore the 7-, 13-, 21-, and 31-day LC50 values were empirically estimated to be >10 mg/L, the highest concentration tested. Consequently, the NOEC was determined to be 10 mg/L and the LOEC was >10 mg/L. All effect parameters are based on mean measured concentrations.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Nov 1998 to 17 Dec 1998

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:

Revised Draft Document April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: For the analysis of the test item concentrations, single samples from the freshly prepared stock solution and duplicate samples from the freshly prepared test media of all test concentrations and the control were taken on Day 0 (first treatment), on Day 12 (second week) and on Day 16 (last week). For the determination of the maintenance of the test item concentrations during the test medium renewal periods, the following additional samples were taken at the end of two treatment periods (on Days 14 and 19):
a) from freshly prepared test media of all test concentrations and the control which were separately incubated during the renewal periods under the same conditions as in the test itself, however without food particles and test animals
b) from the test media of all test concentrations and the control out of the actual test (including the food particles) by pouring together the contents of the test beakers after treatment period.
The first of these two stability control treatments lasted for 48 hours, the second for 72 hours (weekend), corresponding to the two different renewal periods of test media.
- Sample storage conditions before analysis: All samples were deep-frozen (at about - 20 °C) immediately after sampling.

The concentrations of test item were analysed in the duplicate test media samples from the test concentrations of nominal 10 and 22 mg/L, determined in the experiment as the NOEC and the LOEC. From the aged samples, only those containing food particles were analysed. From the control samples only one of the duplicate samples was analysed from each sampling date. The analytical methods and results are described in the attached analytical report.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
At each treatment the highest test concentration of nominal 100 mg/L was freshly prepared by dissolving 0.16 to 0.20 g of the test item homogeneously in 1.6 to 2 L test water by intensive stirring and ultrasonication. Adequate volumes of this intensively stirred test medium were diluted with test water to obtain the additional test concentrations. The test media were always freshly prepared before introduction of the test animals.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Daphnia magna Straus.
- Age at study initiation: Less than 24 hours old and not first brood progeny.
- Method of breeding: Under similar temperature and light conditions as in the test, and in the same kind of test water as used in the test.
- Source: In-house culture
- Feeding during test: The test animals were fed with a food mixture containing one part of green algae of the species Scenedesmus subspicatus (freshly grown in the laboratories of the test facility) and one part of fish food suspension. The fish food suspension was fed in addition to the algal food because a toxic effect of the test item on algae could not be excluded.
- Feeding amount: The carbon contents of the food suspensions was determined. The food amounts were based on the measured concentrations of total organic carbon (TOC) in the food suspensions. The amounts of TOC fed per test animal and day:
Day 0/1: 0.10 mg TOC / Daphnia/ day
Day 2/5-8/12/13: 0.15 mg TOC / Daphnia / day
Day 14-15/19-20: 0.20 mg TOC / Daphnia /day
Day 9/16/19/20: 0.25 mg TOC / Daphnia /day
- Frequency of feeding: On each working day (Monday through Friday)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

20 - 21 °C

pH:

7.0 - 8.1

Dissolved oxygen:

8.2 - 9.0 mg O2/L

Nominal and measured concentrations:

- Nominal concentrations: 0 (blank control), 4.6, 10, 22, 46 and 100 mg/L.
- Mean measured concentrations: 10.4 and 22.4 mg/L for nominal concentrations of 10 and 22 mg/L. 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
- Type: Closed (covered with glass plates)
- Fill volume: 80 mL test medium
- Renewal rate of test solution: Three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

TEST MEDIUM
The test was conducted in reconstituted water (“M7” media) with an initial pH of 7.9 ± 0.3. Before use, the test water was aerated until oxygen saturation. During the test the test media have not been aerated.

WATER PARAMETERS
- pH and oxygen: The pH-values and oxygen concentrations in the test media of all test concentrations and in the control were measured in one beaker each at the beginning and end of each treatment period. Only the pH-values and oxygen concentrations of the lowest and highest test concentration and the control are reported since all pH-values and oxygen concentrations were in the same range.
- Temperature: The water temperature was measured in one control beaker at the start of the test and at the end of each treatment period. Additionally, the air-temperature in the temperature-controlled testing room was continuously recorded.
- Appearance: The appearance of the test media was recorded at each of the above mentioned intervals.

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light and 8-hour darkness
- Light intensity: 300 - 800 Lux

EFFECT PARAMETERS MEASURED
The mortality of the test animals and the number of young were recorded at each test medium renewal date and at the end of the test.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

59 mg/L

95% CI:

> 49 - < 72

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

An overview of the results is provided in Tables 2 - 4 on 'Any other information on results incl. tables'
- Survival: In the control and in all test concentrations all test animals had survived until the end of the test. Thus, the survival rate of Daphnia magna after 21 days was not affected up to a nominal concentration of 100 mg/L.
- Reproduction: The first young offspring released from their parent animals was recorded in the control and at the test concentrations of nominal 4.6 to 46 mg/L at the observation on Day 9. At the highest test concentration (nominal 100 mg/L) a delay of the first brood was determined (first offspring observed on Day 12 of exposure).
- Reproduction rate: The mean reproduction rate of the daphnids in the control was 90 ± 14 alive offspring per adult (mean ± SD). According to the results of a Williams-Test (one-sided smaller, ex. = 0.05) no significant toxic effect of the test item on the mean reproduction rate was determined up to and including the test concentration of nominal 10 mg/L. First at the concentration of nominal 22 mg/L, the mean reproduction rate of surviving daphnids was statistically significantly reduced to in the mean 81% alive offspring compared to the control. The 21-day EC50 for the reproduction rate of Daphnia magna was calculated to be 59 mg/L (95% confidence limits: 49 - 72 mg/L).
- Signs of intoxication: With exception of the reduced reproduction rates, no particular signs of intoxication were observed at the test animals during the test.
- Appearance of the test item in the test media: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Reported statistics and error estimates:

The NOEC and the LOEC of the reproduction rate were evaluated by the multivariate Williams-Test after a one-way analysis of variance (ANOVA).

The EC50 of the reproduction rate at the end of the test was statistically evaluated by Probit Analysis.

Table 2. Number of surviving test animals

Exposure Day	Nominal concentration of the test item (mg/L)
	Control	4.6	10	22	46	100
0	10	10	10	10	10	10
1	10	10	10	10	10	10
2	10	10	10	10	10	10
5	10	10	10	10	10	10
7	10	10	10	10	10	10
9	10	10	10	10	10	10
12	10	10	10	10	10	10
14	10	10	10	10	10	10
16	10	10	10	10	10	10
19	10	10	10	10	10	10
21	10	10	10	10	10	10
% surviving on Day 21	100	100	100	100	100	100

Table 3. Total number of alive, young daphnids reproduced by all adults (cumulative values)

Exposure Day	Nominal concentration  of the test item (mg/L)
	Control	4.6	10	22	46	100
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
5	0	0	0	0	0	0
7	0	0	0	0	0	0
9	119	125	119	105	27	0
12	322	323	309	283	147	83
14	516	582	404	283	259	89
16	616	647	616	537	307	179
19	780	843	806	713	431	257
21	898	918	869	729	567	257
% of control, a.	100.0	102.2	96.8	81.2	63.1	28.6

1. Based on the value of the last exposure day.

Table 4. The number of alive offspring reproduced per surviving adult within 21 days of exposure

Replicate No.	Nominal concentration of the test item (mg/L)
	Control	4.6	10	22	46	100
1	105	119	110	75	53	30
2	111	80	83	77	57	18
3	74	89	78	71	54	29
4	84	88	85	77	57	18
5	79	105	82	75	56	21
6	82	84	112	67	62	23
7	85	88	85	72	77	30
8	93	94	83	70	37	32
9	74	87	81	71	53	27
10	111	84	70	74	61	29
Mean	89.8	91.8	86.9	72.9	56.7	25.7
±SD	14.4	11.7	13.4	3.2	9.9	5.3
n	10	10	10	10	10	10
CV%	16.1	12.8	15.5	4.5	17.5	20.4
Mean in %	100.0	102.2	96.8	81.2	63.1	28.6
STAT	-	n.s.	n.s.	s.	s.	s.

CV %: Coefficient of variation in %: (SDx/meanx)*100%

STAT: Results of a Williams-test with the mean values of alive offspring (one-sided smaller, α = 0.05)

n.s.: mean value not significantly lower than in the control

s.: mean value significantly lower than in the control

Validity criteria fulfilled:

yes

Conclusions:

In a reproduction study on Daphnia magna, performed in accordance with OECD TG 211, the 21-day NOEC for reproduction was determined to be 10 mg/L, based on nominal concentrations.

Executive summary:

The impact of the test item on the reproduction of Daphnia magna was studied in a semi-static system for 21 days. The study was conducted in accordance with OECD TG 211 and in compliance with  GLP criteria. The test organisms (< 24-hours old) were exposed to the test item in reconstituted test water at 20 - 21 ˚C, pH 7.0 – 8.1 and dissolved oxygen 8.0 – 9.0 mg O2/L with a photoperiod of 16 hours daily (300 - 800 Lux). The organisms (1 daphnia/vessel; 10 vessels/concentration or control) were exposed to nominal concentrations of 0 (blank control), 4.6, 10, 22, 46 and 100 mg/L of the test item. The concentrations of the test item were analysed in the duplicate test media samples from the test concentrations of nominal 10 and 22 mg/L using HPLC and UV/VIS-detection. The mean measured concentrations were 10.4 and 22.4 mg/L, respectively. The mortality of the test animals and the number of young were recorded at each test medium renewal date and at the end of the test. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.

The mean measured concentrations of test item in the analysed test media of 10 and 22 mg/L varied in the range of 100 to 112% of the nominal values during the test period. Under the test conditions with food and daphnids, the test item was stable during the test period of 72 hours. Therefore, all reported biological results are related to the nominal concentrations of the test item. Survival of the parental animals was 100% in the control, and in all test concentrations. The first young offspring released from their parent animals was recorded in the control and at the test concentrations of nominal 4.6 to 46 mg/L at the observation on Day 9. At the highest test concentration (nominal 100 mg/L) a delay of the first brood was determined (first offspring observed on Day 12 of exposure). According to the results of a Williams-Test (one-sided smaller, alpha = 0.05) no significant toxic effect of the test item on the mean reproduction rate was determined up to and including the test concentration of nominal 10 mg/L. Based on the findings, the 21-day NOEC and EC50 for reproduction was determined to be 10 and 59 mg/L, respectively, based on nominal concentrations.

Description of key information

Two long-term toxicity studies on aquatic invertebrates are available and included as key studies.

Freshwater, 21-d NOEC = 10 mg/L (based on nominal concentrations), semi-static, reproduction, Daphnia magna, OECD TG 211, Seyfried 1999

Marine water, 31-d NOEC = 10 mg/L (based on mean measured concentrations), flow-through, reproduction, mortality and growth, Americamysis bahia, EPA 850.1350 (draft), Claude 2015

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

10 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

10 mg/L

Additional information

Two studies are available for this endpoint, which were both selected as key studies. Both studies were performed according to standard guidelines and were in compliance with GLP.

Freshwater: Daphnia magna (< 24-hours old) were exposed for 21 days in a semi-static system to the test item at concentrations of 0 (blank control), 4.6, 10, 22, 46 and 100 mg/L. The test organisms (1 daphnia/vessel; 10 vessels/concentration or control) were exposed to the test item in reconstituted test water at 20 - 21 ˚C, pH 7.0 – 8.1 and dissolved oxygen 8.0 – 9.0 mg O2/L with a photoperiod of 16 hours daily (300 - 800 Lux). The concentrations of the test item in the nominal concentrations of 10 and 22 mg/L were analysed and the mean measured concentrations varied between 100 and 112% of the nominal values during the test period. The 21-day NOEC for reproduction was determined to be 10 mg/L, based on nominal concentrations (Seyfried 1999).

Marine water: Americamysis bahia (< 24-hours old) were exposed to the test item for 35 days in a flow-through system to nominal concentrations of 0 (blank control), 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg/L of the test item. The test organisms (initially 15 shrimp/vessel; 4 vessels/concentration or control) were exposed to marine water at 24.7 - 25.9 ˚C, pH 7.8 – 8.1 and dissolved oxygen 6.3 – 7.4 mg O2/L with a salinity of 19-21 ‰ and a photoperiod of 14 hours daily (146 Lux at the start). The mean measured concentrations were 0.33, 0.68, 1.2, 2.5, 5.3 and 10 mg/L, respectively. The NOEC was determined to be 10 mg/L, based on mean measured concentrations (Claude 2015).