Diquat dibromide

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:

11 Dec 2012 to 14 Jan 2013

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, approximately weekly during the test and at test termination to measure concentrations of the test substance. The samples were collected from mid-depth, placed in glass scintillation vials, acidified with two drops of 10% H3PO4 and processed immediately for analysis. Additional samples were collected at test termination as back-up samples and stored refrigerated for possible later analysis.

Vehicle:

no

Details on test solutions:

Individual stock solutions were prepared for each of the concentrations tested, and were prepared five times during the test. For the first two preparations, a primary stock solution of a nominal concentration of 40 mg/mL, correspondent to 8.6 mg/mL test substance cation and 16 mg/mL pure test substance, was prepared by mixing of test material into filtered saltwater . Four secondary stock solutions were prepared in filtered saltwater at nominal concentrations of 2.5, 5.0, 10 and 20 mg/mL, equivalent to 0.54, 1.1, 2.2 and 4.3 mg/mL cation and 1.0, 2.0, 4.0 and 8.0 mg/mL pure test substance, by proportional dilution of the primary stock. In the last three preparations, a primary stock solution was prepared by mixing a calculated amount of test material into filtered saltwater at a nominal concentration of 20 mg/mL. Three secondary stock solutions were prepared in filtered saltwater at nominal concentrations of 2.5, 5.0 and 10 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion. The 2.5 mg/mL stock solution appeared nearly clear and colorless. The 5.0, 10, 20 and 40 mg/mL stock solutions appeared clear and dark brown in color, increasing in intensity with an increase in concentration. No precipitates were visible in any of these stock solutions. Stock solutions were stored refrigerated in glass amber bottles, and aliquots of each stock were placed in the syringe pump every one to three days during the study.

Prior to pairing of the mysids, the five test substance stock solutions were injected into the diluter mixing chambers at a rate of 12.50 µL/minute where they were mixed with dilution water delivered at a rate of 125 mL/minute to achieve the desired test concentrations. Due to 100% mortality that occurred in the 4.0 mg/L (0.86 mg/L cation and 1.6 mg/L test substance) treatment group by Day 8 of the study, toxicant delivery and dilution water flows were ceased to this level following the final analytical sampling interval. Following pairing, the stock solutions were injected into the diluter mixing chambers at a rate of 25.0 µL/minute where they were mixed with dilution water delivered at a rate of 250 mL/minute to achieve the desired test concentrations. The negative control received dilution water only.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid
- Age at study initiation: < 24 hours old
- Source: Obtained as juveniles from cultures maintained at the test facility
- Feeding during test : Fed live brine shrimp nauplii (Artemia sp.) up to four times daily. The mysids in the cultures and test were fed the enriched brine shrimp for one of the daily feedings during the test, when available. The mysid food diet was also periodically supplemented with Skeletonema costatum, a saltwater alga, cultured by the test facility.

ACCLIMATION
- Acclimation period: Adult mysids in the cultures were held in the laboratory for at least 14 days before juveniles were collected for testing.
- Acclimation conditions: During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 25.4 to 25.9 ºC. The pH of the water ranged from 7.8 to 8.4. Dissolved oxygen concentrations were ≥6.8 mg/L (≥ 93% of saturation). Salinity of the filtered saltwater ranged from 19 to 20 ‰.
- Feeding: Fed live brine shrimp nauplii (Artemia sp.) daily. The brine shrimp periodically were enriched with a nutrient enrichment

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

31 d

Test temperature:

25 ± 2 °C

pH:

7.8 - 8.0

Dissolved oxygen:

- 6.9 - 7.4 mg O2/L
- ≥ 94% of saturation

Salinity:

19 – 21‰

Nominal and measured concentrations:

- Nominal concentration: 0 (Negative control), 0.25, 0.50, 1.0, 2.0 and 4.0 mg test material/L, corresponding to 0, 0.10, 0.20, 0.40, 0.84 and 1.5 mg pure test substance/L

- Measured concentration: < LOQ (negative control), 0.24, 0.50, 1.0, 2.1 and 3.7 mg test material/L, respectively. See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
Prior to pairing (Day 0):
- Test vessel: Test compartment placed in each of four replicate test chambers
- Material and size of test vessel: 2 L glass containers measuring approximately 12 cm in diameter and 19 cm in heigh, with two nylon mesh covered holes on opposite sides of the container. The compartments were placed in 9 L glass aquaria containing approximately 2.5 L of test solution. The depth of the water in a representative test chamber and test compartment was 6.6 and 6.6 cm, respectively.
- No. of organisms per vessel: 15
- No. of vessels per concentration: 4
- No. of vessels per control: 4

After sexual maturity (Day 15):
- Test vessel: Reproductive compartments with up to five compartments in each replicate test chamber.
- Material and size of test vessel: The reproductive compartments were approximately 10-cm diameter glass petri dishes with sides of nylon mesh screen. The reproductive compartments were placed in 19 L glass aquaria filled with approximately 14.5 L of test solution, which contained a self-starting siphoning system to exchange test solution. The depth of the water in a representative test chamber and reproductive
compartment was 17.5 and 16.1 cm, respectively.
- No. of organisms per vessel: 2
- No. of vessels per concentration: 5
- No. of vessels per control: 5

TEST APPARATUS
- Apply exposure: The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance and a negative control (dilution water) to test chambers. Syringe pumps were used to deliver test substance stock solutions to impartially assigned mixing chambers where the stocks were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters.A fter mixing, the flow from each mixing chamber was split to deliver test water to four replicate test chambers. The syringe pumps used to deliver stock solutions to the mixing chambers were calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and verified or recalibrated approximately weekly and as needed during the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the four replicates. Delivery of test solutions to the test chambers was initiated one day prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and two times per day during the test.
- Flow rate: Each juvenile test chamber with at least 18 volume additions of test water per day and adult test chambers with at least 6 volume additions of test water per day

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 passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank. The filtered saltwater then was 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 passed through an ultraviolet (UV) sterilizer.

WATER PARAMETERS
In the event of 100% replicate mortality, dissolved oxygen, pH and temperature were taken at that time and then discontinued.
- Temperature: Temperature was measured in each test chamber at the beginning and end of the test, and approximately weekly during the
test, using a hand-held liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber, which was calibrated prior to test initiation and verified or calibrated approximately weekly during the test with a hand-held liquid-in-glass thermometer.
- Dissolved oxygen: Prior to pairing, dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test and approximately weekly during the test period, with measurements rotating among the replicates in each group at each measurement interval. After mysids attained sexual maturity and were paired on Day 15, dissolved oxygen was measured daily until the end of the test in one replicate test chamber of each treatment and 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 control group at the beginning and end of the test, 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
- Photoperiod: 14 hours of light and 10 hours of darkness; 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: 111 lux; Light intensity was measured at the water surface of one representative test chamber at test initiation.

EFFECT PARAMETERS MEASURED:
Observations of the survival (mortality) and behavior of each first-generation mysid were made daily throughout the test. The criteria used to define mortality included lack of movement, absence of respiratory movements, and lack of reaction to gentle prodding. At pairing on Day 15, 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, reproductive females were observed daily for presence of eggs in the brood pouch. 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 test was terminated on Day 31, which was at least seven days past the median time of first brood release for the negative control (Day 23). At test termination, the sex of each surviving first-generation mysid was confirmed and the total length of each mysid was measured using calipers. The mysids then were placed in a drying oven at approximately 60 °C to obtain dry weight data. The mysids were dried for approximately 69 hours.

Reference substance (positive control):

no

Key result

Duration:

31 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

growth

Duration:

31 d

Dose descriptor:

LOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: pure test substance

Basis for effect:

growth

Details on results:

An overview of the results is provided in Table 2 - Table 4 in 'Any other information on results incl. tables'.

Juvenile Survival to Pairing (Days 0 - 15)
Surviving mysids in the control and treatment groups appeared normal during the period from test initiation to pairing on Day15. Observations of organisms in the 0.50 and 1.0 mg/L treatment groups were comparable to those seen in the negative control group. Mysids in the 2.1 and 3.7 mg/L treatment groups exhibited treatment-related signs of toxicity including an increased number of small organisms relative to the negative control group, lethargy and mortality. After 15 days of exposure, survival of juvenile mysids in the negative control and in the 0.24, 0.50, 1.0, 2.1 and 3.7 mg/L treatment groups was 98.3, 95.0, 96.7, 95.0, 80.0 and 0.0%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 2.1 and 3.7 mg/L treatment groups when compared to the negative control (p ≤ 0.05). The marked decrease in survival among mysids in these two treatment groups was considered to be treatment related. Consequently, the LOEC for juvenile survival (Days 0 – 15) was 2.1 mg/L, and the NOEC was 1.0 mg/L.

Adult Survival After Pairing (Days 16 - 31)
In general, surviving mysids in the 0.24 to 2.1 mg/L treatment groups appeared normal during this period. There were a few observations of organisms that appeared small or exhibited lethargy, however, these observations were infrequent and were not considered to be treatment related. At test termination, survival of adult mysids in the negative control and in the 0.24, 0.50, 1.0 and 2.1 mg/L (0.052, 0.11, 0.22 and 0.45 mg/L cation and 0.10, 0.20, 0.40 and 0.84 mg/L active ingredient) treatment groups was 88.9, 80.0, 69.8, 84.8 and 90.9%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 0.50 mg/L (0.11 mg/L cation and 0.20 mg/L active ingredient) treatment group when compared to the negative control (p ≤ 0.05). While statistically significant, the decrease in survival in the 0.50 mg/L (0.11 mg/L cation and 0.20 mg/L active ingredient) treatment group was not considered to be treatment related, since the decrease was slight and was not dose-responsive. Consequently, the NOEC for adult survival (Days 16 – 31) was 2.1 mg/L (0.45 mg/L cation and 0.84 mg/L active ingredient), the highest concentration tested during the adult phase of the study.

Reproduction
For each female, the number of reproductive days was defined as the number of days that the female was alive from the day of first brood release of any female in the test to the end of the test. The day of first brood release in this study was Day 16. The mean percent of surviving females producing young in the negative control and in the 0.24, 0.50, 1.0 and 2.1 mg/L (0.052, 0.11, 0.22 and 0.45 mg/L cation and 0.10, 0.20, 0.40 and 0.84 mg/L active ingredient) treatment groups was 100, 100, 100, 100 and 94.4%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in mean percent of surviving females producing 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 negative control and the 0.24, 0.50, 1.0 and 2.1 mg/L (0.052, 0.11, 0.22 and 0.45 mg/L cation and 0.10, 0.20, 0.40 and 0.84 mg/L active ingredient) treatment groups was 13.2, 10.8, 12.3, 10.8 and 11.0, 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).
Both of these endpoints were 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. Control reproductive performance in this study met the ASTM Standard E 1191-03a Guideline acceptability criteria, which states that at least 75% of the first-generation females in the control(s) must produce young and that the average be at least three young produced per first-generation female.
The mean number of young produced per reproductive day in the negative control and in the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 0.826, 0.699, 0.640, 0.674 and 0.672, 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).
Observations of second-generation mysids revealed no abnormal development or abnormal behavior, except for an occasional lethargic organism, in any of the treatment or control groups.

Growth (Males)
The mean total length and dry weight of male mysids in the negative control group was 8.27 mm and 1.10 mg, respectively. The mean total length of male mysids in the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 8.27, 8.29, 8.28 and 8.07 mm, respectively. The mean dry weight of males in 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 1.05, 1.06, 1.02 and 0.97 mg, respectively. Dunnett’s test indicated there were statistically significant decreases in mean dry weight for males in the 2.1 mg/L treatment group, in comparison to the negative control (p ≤ 0.05).

Growth (Females)
The mean total length and dry weight of female mysids in the negative control group was 8.52 mm and 1.41 mg, respectively. The mean total length of female mysids in the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 8.24, 8.35, 8.36 and 8.25 mm, respectively. The mean dry weight of females in the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 1.28, 1.17, 1.20 and 1.10 mg, respectively. Dunnett’s test indicated there was a statistically significant decrease in mean dry weight for females in the 0.50, 1.0 and 2.1 mg/L treatment groups, in comparison to the negative control (p > 0.05).
Based on the effects on growth observed in the 0.50, 1.0 and 2.1 mg/L treatment groups, the LOEC for growth (female dry weight) was 0.50 mg/L, equivalent to 0.20 mg/L pure test substance, and the NOEC was 0.24 mg/L, equivalent to 0.10 mg/L pure test substance.

Reported statistics and error estimates:

See 'Statistical analysis' in 'Any other information on materials and methods incl. tables'.

Table 2. Summary of survival of saltwater mysids exposed to the test substance

Survival of Saltwater Mysids
Mean Measured Concentration (mg/L)			Juvenile Survival to			Adult Survival to Test
			Pairing on Day 15			Termination on Day 31
Test material (mg/L)	test substance cation (mg/L)	test substance equivalent (mg/L)	Number Originally Exposed	Number Surviving	Percent Survival	Number Alive at Pairing^1	Number Surviving	Percent Survival
Negative Control	Negative Control	Negative Control	60	59	98.3	54	48	88.9
0.24	0.052	0.10	60	57	95.0	45	36	80.0
0.50	0.11	0.20	60	58	96.7	43	30	69.8*^2
1.0	0.22	0.40	60	57	95.0	46	39	84.8
2.1	0.45	0.84	60	48	80.0*	44	40	90.9
3.7	0.80	1.5	60	0	0.0*	--^3	--^3	--^3

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

1 The number alive at pairing may be less than the number surviving to Day 15 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 15.

2 While the decrease in survival was statistically significant in comparison to the negative control, it was not considered to be treatment-related since the difference was not dose-responsive.

3 The 3.7 mg/L treatment group reached 100% mortality during the juvenile phase of the study

 

 

Table 3. Summary of reproduction of saltwater mysids exposed to the test substance

Mean Measured Concentration (mg/L)			Mean Number of Young Produced Per Reproductive Day ± SD 1	Percent of Surviving Females Producing Young ± SD 2,3	Mean Number of Young Per Surviving Female ± SD 1,3
Test material (mg/L)	test substance cation (mg/L)	test substance (mg/L)
Negative Control	Negative Control	Negative Control	0.826 ± 0.127	100	13.2 ± 2.01
0.24	0.052	0.10	0.699 ± 0.096	100	10.8 ± 0.92
0.50	0.11	0.20	0.640 ± 0.148	100	12.3 ± 3.11
1.0	0.22	0.40	0.674 ± 0.153	100	10.8 ± 3.08
2.1	0.45	0.84	0.672 ± 0.237	94.4	11.0 ± 3.65

1 There were no statistically significant decreases in reproduction and average number of young per surviving female in comparison to the negative control using Dunnett’s test (p > 0.05).

2 There were no statistically significant decreases in percent of females producing young in comparison to the negative control using Fisher’s Exact test (p > 0.05).

3 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 4. Summary of growth of saltwater mysids exposed to the test substance

Mean Measured Concentration (mg/L)			Growth Parameters at Termination on Day 31
Test material (mg/L)	test substance cation (mg/L)	test substance  (mg/L)	Mean Total Length ± SD (mm)		Mean Dry Weight ± SD (mg)
			Males	Females	Males	Females
Negative Control	Negative Control	Negative Control	8.27 ± 0.108	8.52 ± 0.124	1.10 ± 0.017	1.41 ± 0.013
0.24	0.052	0.10	8.27 ± 0.186	8.24 ± 0.169	1.05 ± 0.079	1.28 ± 0.110
0.50	0.11	0.20	8.29 ± 0.172	8.35 ± 0.139	1.06 ± 0.089	1.17 ± 0.038*
1.0	0.22	0.40	8.28 ± 0.050	8.36 ± 0.484	1.02 ± 0.038	1.20 ± 0.163*
2.1	0.45	0.84	8.07 ± 0.220	8.25 ± 0.189	0.97 ± 0.069*	1.10 ± 0.103*

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

 

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 31-day NOEC for growth was determined to be 0.10 mg test substance/L and the LOEC (female dry weight) was 0.20 mg test substance/L.

Executive summary:

To evaluate the effects of the test substance on the survival, reproduction and growth of the saltwater mysid (Americamysis bahia), the organisms (< 24 hours old) were exposed to a geometric series of five test concentrations and a negative control (dilution water) under flow-through conditions for 31 days. The study was conduced in accordance with EPA 850.1350 guideline and in compliance with GLP criteria.

The mean concentrations measured by HPLC/UV were 0.24, 0.50, 1.0, 2.1 and 3.7 mg test material/L, respectively, corresponding to 0.10, 0.20, 0.40, 0.84 and 1.5 mg pure test substance/L. At test initiation, each replicate contained one compartment with 15 neonate mysids, resulting in a total of 60 mysids in each treatment and control group. On Day 15 of the test, after mysids attained sexual maturity, males and females were paired in each treatment and control group, with a maximum of five reproductive pairs per replicate. Reproduction of the paired mysids was monitored through termination on Day 31. Observations for mortality and signs of toxicity were conducted daily throughout the test. At test termination, the total body lengths and dry weights of all surviving first-generation mysids were measured. The study was carried out in 25 ± 2 °C water temperature, with approximately 6.9 mg O2/L dissolved oxygen concentrations (remained ≥ 94% of saturation), pH 7.8 - 8.0, and salinity 19 - 21‰. Light intensity was 111 lux at the surface of the water of one representative test chamber.

 

Surviving mysids in the control and treatment groups appeared normal during the period from test initiation to pairing on Day15. Observations of organisms in the 0.50 and 1.0 mg/L treatment groups were comparable to those seen in the negative control group. Mysids in the 2.1 and 3.7 mg/L treatment groups exhibited treatment-related signs of toxicity including an increased number of small organisms relative to the negative control group, lethargy and mortality. After 15 days of exposure, survival of juvenile mysids in the negative control and in the 0.24, 0.50, 1.0, 2.1 and 3.7 mg/L treatment groups was 98.3, 95.0, 96.7, 95.0, 80.0 and 0.0%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 2.1 and 3.7 mg/L treatment groups when compared to the negative control (p ≤ 0.05). The marked decrease in survival among mysids in these two treatment groups was considered to be treatment related. Consequently, the LOEC for juvenile survival (Days 0 – 15) was 2.1 mg test material/L, equivalent to 0.84 mg/L pure test substance. The NOEC was 1.0 mg/L, corresponding to 0.40 mg/L test substance.

 

In general, surviving mysids in the 0.24 to 2.1 mg/L treatment groups appeared normal during this period. There were a few observations of organisms that appeared small or exhibited lethargy, however, these observations were infrequent and were not considered to be treatment related. At test termination, survival of adult mysids in the negative control and in the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 88.9, 80.0, 69.8, 84.8 and 90.9%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 0.50 mg/L treatment group when compared to the negative control (p ≤ 0.05). While statistically significant, this decrease in survival was not considered to be treatment related, since the it was slight and not dose-responsive. 

The mean percent of surviving females producing young in the negative control and in the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 100, 100, 100, 100 and 94.4%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in mean percent of surviving females producing 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 negative control and the 0.24, 0.50, 1.0 and 2.1 mg/L treatment groups was 13.2, 10.8, 12.3, 10.8 and 11.0, 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). 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). 

The guideline acceptability criteria, that at least 75% of the first-generation females in the control(s) must produce young and that the average be at least three young produced per first-generation female, were met.

Observations of second-generation mysids revealed no abnormal development or abnormal behavior, except for an occasional lethargic organism, in any of the treatment or control groups. The mean total length of male mysids was not affected by treatment, but there were statistically significant decreases in mean dry weight for males in the 2.1 mg/L treatment group, in comparison to the negative control (p ≤0.05).

The mean total length and dry weight of female mysids in the negative control group was 8.52 mm and 1.41 mg, respectively. Likewise, the total length of female mysids was not affected by exposure to the substance, but there was a statistically significant decrease in mean dry weight for females in the 0.50, 1.0 and 2.1 mg/L treatment groups, in comparison to the negative control (p > 0.05).

Based on the effects on the growth of male and femaly mysids, the 31-day LOEC for growth (female dry weight) was 0.50 mg test material/L, corresponding to 0.20 mg pure test substance/L. The NOEC was 0.24 mg test material/L, corresponding to 0.10 mg pure test substance/L.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1990 to Jun 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

OECD TG 202 - part II (1984)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All of the freshly prepared test solutions and the old test solutions on days 2, 5 , 12, 19 and 21 were analysed for the cation.

Vehicle:

no

Details on test solutions:

The top concentration was prepared by adding the test substance technical directly to the dilution water to give a concentration of 0.5 mg cation/L and lower concentrations from this by serial dilution. The control was dilution water only.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: From cultures maintained at the test facility
- Age at study initiation: < 24 hours old (produced from females from a single culture vessel)
- Feeding during test: The Daphnia were fed daily with suspensions of Chlorella vulgaris(4E+07 cells per vessel) and dried yeast (0.25 mg per vessel).

ACCLIMATION
- Acclimation period: 16 days
- Acclimation conditions: 20 °C
- Type and amount of food: Yeast and Chlorella vulgaris

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

165 mg/L as CaCO3

Test temperature:

20 ± 1 °C

pH:

7.6 - 8.8

Dissolved oxygen:

≥ 7.5 mg O2/L

Conductivity:

335 - 365 µS/cm

Nominal and measured concentrations:

- Nominal concentration: 0, 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg test substance cation/L
- Measured concentration:

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL glass beaker
- Fill volume: 50 mL of test solution
- Type: Closed (covered with a watch glass to reduce evaporation)
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control (replicates): 10

TEST MEDIUM
- Source/preparation of dilution water: The dilution water used was of hardness 160 - 180 mg/L (as CaCO3). The water is produced by mixing mains, dechlorinated water (hardness approximately 300 mg/L as CaCO3) with deionised water to give the required hardness.

WATER PARAMETERS
- Dissolved oxygen and pH: Dissolved oxygen and pH were measured in one replicate at each test concentration from the freshly prepared test solutions on study days 0, 2, 5, 12 and 19. Measurements were also taken from a single replicate of the old solutions on study days 2, 5, 12, 19 and 21.
- Temperature: The temperature of the constant temperature room was recorded continuously using a thermohygrograph.
- Alkalinity, conductivity and hardness: Water samples were taken of the blended water used to prepare the test solutions for analysis of alkalinity and specific conductivity and study days 2,9 and 19 and for total hardness on days 2, 10 and 16.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 700 lux

EFFECT PARAMETERS MEASURED
At the start of the study a single Daphnia was added to each test vessel. These were termed the P0 generation. Mortality of the P0 generation was recorded daily for each test vessel. Fresh test solutions were prepared every Monday, Wednesday and Friday. Surviving P0 generation were transferred to the new solutions using a wide bore pipette. Old test solutions were examined, from study day 7, for young Daphnia (termed the F1 generation). Any offspring present, both live and dead, were couted an dthen discarded. At the end of the test (day 21) the length (from apex of helmet to base of spine) of the surviving P0 Daphnia was measured, using a microscope with a calibrated eyepiece graticule.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.234 mg/L

Nominal / measured:

nominal

Conc. based on:

other: pure test substance

Basis for effect:

other: mortality, growth (adult length) and the number of produced young per daphnia

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.125 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

other: mortality, growth (adult length) and the number of produced young per daphnia

Remarks on result:

other: Original value presented in study

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

other: mortality, growth (adult length) and the number of produced young per daphnia

Remarks on result:

other: Original value presented in study

Details on results:

Any overview of the results in provided in Table 2 to Table 4 in ‘Any other information on results incl. Tables’.

- Survival: At 0.125 mg cation/L, concentrations below this and in the controls at least 90% of the daphnia survived. 100% mortality was observed on after 10 days exposure to 0.25 mg cation/L or 0.5 mg cation/L treatments.
- Growth: The mean length of daphnia were 4.66, 4.58, 4.60 and 4.65 mm in the control, 0.03125, 0.0625, 0.0125 mg cation/L treatment groups, respectively. There were no significant differences in the mean adult Daphnia length on day 21, between those at 0.125 mg cation/L, those in lower concentrations and the control.
- Total number of young produced per daphnia: At nominally 0.5 and 0.25 mg cation/L, there were no young. At concentrations below this the mean number of young produced was not significantly different to the control. The results for numbers of young produced at 0.0625 mg cation/L were analysed both with and without replicate. This replicate did not produce any young (even though the daphnia was female), however, it is thought unlikely that this was a treatment related effect. The results of both analyses showed no significant difference to the control.
The 21 day NOEC based on nominal concentration, was therefore 0.125 mg test substance cation/L, and the LOEC was 0.25 mg test substance cation/L.

Reported statistics and error estimates:

- Mortality: Estimates of the LC50 and its 95% confidence Intervals on days 7, 14 and 21 were produced from analysing the data by iteratively reweighted linear regression of the probit transformation of % mortality on log (nominal concentration).
- Number of Young and Daphnia Length: Number of young produced (F1 generation) and Daphnia length (P0 generation), day 21, were analysed for significant differences from the controls. Prior to analysis, numbers of young were logarithmically transformed. Parent length was not transformed. Each of these variables was then analysed by a one-way analysis of variance, using all treatments, to obtain an estimate of experimental error. Using this, the least significant difference(LSD) was computed at the 5% and 1% probability levels in order to determine if there were any significant differences between the treated groups and the control.

Table 2. Mortality

Replicate	Study Day Daphnia assessed dead
	Nominal Concentration (mg test substance cation/L)
	Control	0.03125	0.0625	0.125	0.25	0.5
1	+	+	+	+	8	5
2	+	+	+	+	7	5
3	+	+	+	+	7	5
4	+	+	17	+	7	3
5	+	+	+	+	7	5
6	+	+	+	+	10	4
7	+	+	+	+	7	4
8	+	+	+	+	7	4
9	+	+	+	+	7	4
10	+	+	+	+	7	4
Total dead^b
Day 7	0	0	0	0	8	10
Day 14	0	0	0	0	10	10
Day 21	0	0	1	0	10	10

a. + indicates Daphnia surviving at Day 21

b. data used to calculate LC50 values

 

Table 3. Daphnia Lengths, Day 21

Replicate	Length in mm
	Nominal concentration (mg test substance cation /L)
	Control	0.03125	0.0625	0.125
1	4.70	4.51	3.81	4.57
2	4.76	4.38	4.76	4.44
3	4.63	4.63	4.63	4.44
4	4.76	4.44	-^b	4.83
5	4.57	4.95	4.70	4.63
6	4.44	4.76	4.76	4.70
7	4.70	4.70	4.76	4.76
8	4.70	4.57	4.44	4.83
9	4.76	4.19	4.70	4.76
10	4.57	4.70	4.83	4.57
Mean^c	4.66	4.58	4.6	4.65

 LSD 5% = 0.191

LSD 1% - 0.257

a. All Daphnia at nominal concentrations 0.25 and 0.5 mg cation/L were dead before day 21

b. - Daphnia dead before day 21

c. Means not significantly different

Table 4. Total Number of Young Produced per Daphnia

Replicate	Total Number of Live Young
	Nominal Concentration (mg test substance cation/L)
	Control	0.03125	0.0625	0.125	0.25	0.5
1	121	123	0	130	0	0
2	127	113	134	139	0	0
3	137	127	154	118	0	0
4	126	113	126	134	0	0
5	110	174	160	153	0	0
6	107	119	114	149	0	0
7	120	131	152	138	0	0
8	121	121	137	116	0	0
9	116	127	132	132	0	0
10	157	106	141	186	0	0
Mean	124	125	139^a(125)^b	140	0	0
Detransformed mean	126c	126^c	140^a,c(86)^b,c	140^c	0	0

Figures in brackets are means including.

a. mean excluding replicate 1

b. mean including replicate 1

c detransformed means are not significantly different.

 

Calculation of key result

The original effect levels were expressed as cation species of the test substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of test substance divided by 184.2 g/mol molecular weight of cation species):

1.868 x 0.125 mg/L = 0.234 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 21 day NOEC based on nominal concentration was therefore 0.125 mg test substance cation/L, equivalent to 0.234 mg pure test substance/L. The LOEC was 0.25 mg test substance cation/L, corresponding to 0.47 mg pure test substance/L.

Executive summary:

To investigate the chronic effect on aquatic invertebrates, Daphnia magna were exposed to a series of concentrations of the test substance in water and an untreated control at 20 ± 1 °C. The study was conducted according to OECD TG 202 part II (1984), which is similar to OECD 211, and in compliance with GLP criteria. The nominal concentration were 0 (Negative control), 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg test substance cation/L. The concentrations measured by HPLC confirmed that these concentrations were achieved and all effect levels were therefore based on nominal test concentrations. Each test concentration consisted of 10 replicate chambers, originally containing a single daphnia. Daphnids were transfered to freshly prepared test solutions three times weekly. The pH was in a range of 7.6 – 8.8 and the dissolved oxygen concentration was ≥ 7.5 mg O2/L during the test.

All daphnids in the control, 0.03125 mg cation/L and 0.125 mg cation/L treated groups survived the 21 days exposure. One daphnia was found dead in 0.0625 mg cation/L treatment on day 21. 100% mortality was observed on day 14 at 0.25 mg cation/L treatment and on day 7 at 0.5 cation/L treatment. There were no significant differences in the mean adult daphnia length on day 21, between the daphnids in the control and treatment groups of 0.125 mg cation/L and lower concentrations. At 0.5 and 0.25 mg cation/L, there were no young produced and all daphnia were dead by day 10. At concentrations below this the mean number of young produced was not significantly different to the control. One replicate at 0.0625 mg/L did not produce any young, even though the daphnia was female, however, it is thought unlikely that this was a treatment related effect. The results of analyses including and excluding this replicate showed no significant difference to the control.

Based on the findings, the 21-day NOEC based on nominal concentration was therefore 0.125 mg test substance cation/L, equivalent to 0.234 mg test substance/L. The LOEC was 0.25 mg test sbustance cation/L, corresponding to 0.47 mg pure test substance/L.

Description of key information

Freshwater, 21-d NOEC = 0.234 mg registered substance/L, mortality, growth (length) and number of produced young per daphnia, Daphnia magna, similar to OECD 211, Rapley et.al, 1991

Marine water, 31-d NOEC = 0.10 mg registered substance/L, growth, Americamysis bahia, EPA 850.1350, Claude 2013

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.234 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.1 mg/L

Additional information

Freshwater

To investigate the chronic effect on aquatic invertebrates, Daphnia magna were exposed to a series of concentrations of the test substance in water and an untreated control at 20 ± 1 °C. The study was conducted according to OECD TG 202 part II (1984), which is similar to OECD 211, and in compliance with GLP criteria. The nominal concentration were 0 (Negative control), 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg test substance cation/L. The concentrations measured by HPLC confirmed that these concentrations were achieved and all effect levels were therefore based on nominal test concentrations. Each test concentration consisted of 10 replicate chambers, originally containing a single daphnia. Daphnids were transfered to freshly prepared test solutions three times weekly. The pH was in a range of 7.6 – 8.8 and the dissolved oxygen concentration was ≥ 7.5 mg O2/L during the test.

All daphnids in the control, 0.03125 mg cation/L and 0.125 mg cation/L treated groups survived the 21 days exposure. One daphnia was found dead in 0.0625 mg cation/L treatment on day 21. 100% mortality was observed on day 14 at 0.25 mg cation/L treatment and on day 7 at 0.5 cation/L treatment. There were no significant differences in the mean adult daphnia length on day 21, between the daphnids in the control and treatment groups of 0.125 mg cation/L and lower concentrations. At 0.5 and 0.25 mg cation/L, there were no young produced and all daphnia were dead by day 10. At concentrations below this the mean number of young produced was not significantly different to the control. One replicate at 0.0625 mg/L did not produce any young, even though the daphnia was female, however, it is thought unlikely that this was a treatment related effect. The results of analyses including and excluding this replicate showed no significant difference to the control.

Based on the findings, the 21-day NOEC based on nominal concentration was therefore 0.125 mg test substance cation/L, equivalent to 0.234 mg test substance/L. The LOEC was 0.25 mg test sbustance cation/L, corresponding to 0.47 mg pure test substance/L.

 

Marine water

To evaluate the effects of the test substance on the survival, reproduction and growth of the saltwater mysid (Americamysis bahia), the organisms (< 24 hours old) were exposed to a geometric series of five test concentrations and a negative control (dilution water) under flow-through conditions for 31 days. The study was conduced in accordance with EPA 850.1350 guideline and in compliance with GLP criteria.

The mean concentrations measured by HPLC/UV were 0.24, 0.50, 1.0, 2.1 and 3.7 mg test material/L, respectively, corresponding to 0.10, 0.20, 0.40, 0.84 and 1.5 mg pure test substance/L. At test initiation, each replicate contained one compartment with 15 neonate mysids, resulting in a total of 60 mysids in each treatment and control group. On Day 15 of the test, after mysids attained sexual maturity, males and females were paired in each treatment and control group, with a maximum of five reproductive pairs per replicate. Reproduction of the paired mysids was monitored through termination on Day 31. Observations for mortality and signs of toxicity were conducted daily throughout the test. At test termination, the total body lengths and dry weights of all surviving first-generation mysids were measured. The study was carried out at 25 ± 2 °C water temperature, with approximately 6.9 mg O2/L dissolved oxygen concentrations (remained ≥ 94% of saturation), pH 7.8 - 8.0, and salinity 19 - 21‰. Light intensity was 111 lux at the surface of the water of one representative test chamber.

Surviving mysids in the control and treatment groups appeared normal during the period from test initiation to pairing on Day15. Mysids in the 2.1 and 3.7 mg/L treatment groups exhibited treatment-related signs of toxicity including an increased number of small organisms relative to the negative control group, lethargy and mortality. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 2.1 and 3.7 mg/L treatment groups when compared to the negative control (p ≤ 0.05). The marked decrease in survival among mysids in these two treatment groups was considered to be treatment related. 

At test termination, survival of adult mysids in the 0.50 mg/L treatment group was decreased when compared to the negative control (p ≤ 0.05). While statistically significant, this decrease in survival was not considered to be treatment related, since the it was slight and not dose-responsive, as the survival of adults at higher exposure was unaffected by treatment. 

The number of reproductive days was defined as the number of days that a female was alive from the day of first brood release of any female, which was Day 16 in the test, to the end of the test. There were no statistically significant decreases in mean percent of surviving females producing young in any of the treatment groups, in comparison to the negative control (p >0.05). There were no statistically significant decreases in the mean number of young produced per surviving female and per reproductive day in any of the treatment groups when compared to the negative control (p >0.05).  

The guideline acceptability criteria, that at least 75% of the first-generation females in the control(s) must produce young and that the average be at least three young produced per first-generation female, were met.

Observations of second-generation mysids revealed no abnormal development or abnormal behavior, except for an occasional lethargic organism, in any of the treatment or control groups. The mean total length of male mysids was not affected by treatment, but there were statistically significant decreases in mean dry weight for males in the 2.1 mg/L treatment group, in comparison to the negative control (p ≤0.05).

The mean total length and dry weight of female mysids in the negative control group was 8.52 mm and 1.41 mg, respectively. Likewise, the total length of female mysids was not affected by exposure to the substance, but there was a statistically significant decrease in mean dry weight for females in the 0.50, 1.0 and 2.1 mg/L treatment groups, in comparison to the negative control (p > 0.05).

Based on the effects on the growth of male and femaly mysids, the 31-day LOEC for growth (female dry weight) was 0.50 mg test material/L, corresponding to 0.20 mg pure test substance/L. The NOEC was 0.24 mg test material/L, corresponding to 0.10 mg pure test substance/L.