Paraquat-dichloride

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:

21 Oct 2013 to 27 Nov 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

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E 1191-03a: Standard Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids

Version / remarks:

2008

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Prior to initiation of the exposure, analytical chemistry confirmed that the diluter exposure system was functioning properly and was adequately equilibrated. 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 test termination to measure concentrations of the test substance. The samples were collected from mid-depth, placed in plastic vials, and one drop of formic acid was added to each vial. The samples were processed immediately for analysis. An additional set of samples was collected at test termination and stored refrigerated for possible future analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Individual stock solutions were prepared for each of the six concentrations tested. A primary stock solution was prepared by mixing a calculated amount of test substance into reverse osmosis water at a nominal concentration of 4000 μg technical test substance/mL. Five secondary stock solutions were prepared in R.O. water at nominal concentrations of 130, 250, 500, 1000 and 2000 μg technical test substance/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and ranged in appearance from clear and colorless to clear and brown. Stock solutions were stored ambient in glass amber bottles, and aliquots of each stock were placed in the syringe pump every one to three days during the exposure period. 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. 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 shrimp
- Source: Wildlife International cultures
- Age: Mysids used in the test were obtained as juveniles (<24 hours old).
- Feeding: Mysids in the cultures were fed live brine shrimp nauplii (Artemia sp.) daily. The brine shrimp were enriched daily with a nutrient enrichment. During the test, the mysids were fed live brine shrimp nauplii (Artemia sp.) up to four times daily. 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 cultures and test were fed the enriched brine shrimp for one of the daily feedings during the test. Mysid food was also supplemented daily with Skeletonema costatum, a saltwater alga. Excess food and waste were siphoned out daily during observations.

ACCLIMATION
- Acclimation period: Adult mysids in the cultures were held in the laboratory for at least 14 days before neonates were collected for testing.
- Acclimation conditions: 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 25.7 to 26.1ºC, measured with a liquid-in-glass thermometer. The pH of the water ranged from 8.1 to 8.3, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥6.8 mg/L (≥93% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter. Salinity of the filtered saltwater was 20 parts per thousand (‰), measured with a VitalSine refractometer.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Remarks on exposure duration:

First generation (G1) were exposed to 28 days, second generation (G2) saltwater mysids were evaluated for survival for 96 hours

Test temperature:

25.3 - 26.5 °C; temperature monitored continuously in the negative control replicate A test chamber during the test ranged from approximately 24.92 to 27.15ºC, measured to the nearest 0.01 ºC.

pH:

7.9 - 8.0

Dissolved oxygen:

6.7 - 7.4 mg O2/L; a dissolved oxygen concentration of 4.4 mg/L represents 60% saturation at 25ºC in saltwater with a salinity of 20‰. A dissolved oxygen concentration of 7.4 mg/L represents 100% saturation at 25ºC in saltwater with a salinity of 20‰.

Salinity:

19 - 20 ‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 13, 25, 50, 100, 200 and 400 μg technical test substance/L
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Pre-pairing test vessel: Prior to pairing, mysids in each treatment and control group were held in one test compartment placed in each of four replicate test chambers. The compartments were 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 5.8 and 5.5 cm, respectively.
- Pairing test vessel: After mysids attained sexual maturity and were paired on Day 14, 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 μmg 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 15.1 and 14.8 cm, respectively. An additional compartment was maintained in each test chamber to house any G2 mysids for a 96-hour evaluation period, when available.
- Type of flow-through: 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 negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, South Natick, Massachusetts) 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, and was adjusted to provide 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. After 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 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 three days 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 at least two times per day during the test.
- Introduction of individuals: To initiate the exposure, the juvenile mysids (<24 hours old) were collected and were impartially distributed one to three at a time into transfer containers until each container held 15 mysids. Each group of mysids then was transferred to an indiscriminately assigned test compartment. All transfers were performed using wide-bore pipettes below the water surface.
- No. of organisms per pre-paring vessel: 15
- No. of pairs per paring vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- 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 37800-L storage tank. The filtered saltwater was then diluted to a salinity of approximately 20‰ with freshwater from a well on the Wildlife International site and was aerated with spray nozzles. Prior to use, the 20‰ water was filtered to 0.45 μm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Culture medium different from test medium: Same

WATER QUALITY PARAMETERS
- Temperature: The target test temperature during the study was 25 ± 2°C. 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 using a Fulscope ER/C Recorder and a validated environmental monitoring system (Amegaview Central Monitoring System), which were calibrated prior to exposure 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 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 14, 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. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter.
- 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 test, and approximately weekly during the test, with measurements rotating among the replicates in each group at each measurement interval. Measurements of pH were made using a Thermo Orion Model 525Aplus meter.
- 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. Salinity was measured using a VitalSine refractometer.

OTHER TEST CONDITIONS
- Photoperiod: Lighting was controlled by an automatic timer to provide a photoperiod of 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.
- Lighting type: Ambient laboratory light was used to illuminate the exposure systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: 115 lux; light intensity was measured at the water surface of one representative test chamber at exposure initiation using a SPER Scientific Model 840006C light meter.

EFFECT PARAMETERS MEASURED: G1 mortality, sublethal effects, reproduction, length, dry weight, offspring (G2) mortality, sublethal effects
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 14, 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 28, which was at least seven days past the median time of first brood release for the negative control (Day 19). 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 71 hours. After drying, the mysids were then measured for dry weight to 0.01 mg for data collection.

When available, one group per replicate in each control and test concentration of G2 mysids (n≤10) produced during the reproductive phase were maintained under their respective test conditions and observed for mortality, signs of toxicity, appearance, and behavior for 96 hours post release. The G2 mysids were collected preferentially from multiple G1 pairs within each replicate to obtain up to 10 offspring per replicate at a time. When necessary, offspring produced on the same day were collected and pooled from different replicates per concentration to obtain the required number of animals.

RANGE-FINDING STUDY
Nominal test concentrations were selected based on the results of exploratory range finding toxicity data (not further reported).

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

52.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: pure test substance

Basis for effect:

reproduction

Remarks on result:

other: recalculated value expressed as pure substance, see 'Any other information on results incl. tables' for respective calculation

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

113 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: original value presented in the study (technical test item)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

105.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: pure test substance

Basis for effect:

reproduction

Remarks on result:

other: recalculated value, expressed as pure substance, see 'Any other information on results incl. tables' for respective calculation

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

228 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: original value presented in the study (technical test item)

Details on results:

A detailed overview of the results and all derived effect values are presented in 'Any other information on results incl. tables'.

G1 JUVENILE SURVIVAL TO PAIRING (DAY 0 - 14)
Surviving mysids in the control and treatment groups appeared normal during the period from exposure initiation to pairing on Day 14. There were a few observations of organisms in the 15 to 113 μg technical test substance/L treatment groups that appeared small or lethargic. These observations were infrequent and not concentration-responsive, therefore, they were not considered to be treatment-related. In the 228 and 457 μg technical test substance/L treatment groups, the observations or lethargy, relative size compared to the control and erratic swimming were considered to be treatment related effects. After 14 days of exposure, survival of juvenile mysids in the negative control was 96.7% survival of juvenile mysids in the 15, 28, 58, 113, 228 and 457 μg technical test substance technical/L treatment groups was 91.7, 91.7, 100, 96.7, 90.0 and 60.0%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 457 μg technical test substance/L treatment group when compared to the negative control (p ≤ 0.05).

G1 ADULT SURVIVAL AFTER PAIRING (DAY 15 - 28)
Surviving mysids in the negative control and the treatment groups appeared normal during this period. There were a few observations of organisms that exhibited lethargy and erratic swimming, however, these observations were infrequent and were not considered to be treatment related. At test termination, survival of adult mysids in the negative control was 79.2%. Survival of adult mysids in the 15, 28, 58, 113, 228 and 457 μg technical test substance technical/L treatment groups was 91.9, 81.0, 75.5, 74.4, 48.8 and 32.1%, respectively. Fisher’s Exact test indicated there was a statistically significant decrease in survival in the 228 and 457 μg technical test substance/L treatment groups when compared to the negative control (p ≤ 0.05). There was less than 50% mortality in any of the treatment groups tested through Day 14 of the study.

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, 5.5 and 0.352, respectively. The percent of surviving females producing young in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 76.5, 76.5, 94.4, 78.6, 64.3 and 0.0%, respectively. Fisher’s Exact test indicated there were statistically significant decreases in percent of females reproducing young in the 228 and 457 μg technical test substance/L treatment groups, in comparison to the negative control (p ≤ 0.05). The mean number of young produced per surviving female in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 4.9, 5.4, 4.0, 6.8, 2.5 and 0.0, respectively. Dunnett’s test and Jonckheere-Terpstra trend test indicated there were statistically significant decreases in the mean number of young produced per female in the 457 μg technical test substance/L treatment group when compared to the negative control (p ≤ 0.05). The mean number of young produced per reproductive day in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 0.346, 0.374, 0.285, 0.479, 0.170 and 0.000, respectively. Dunnett’s test and Jonckheere-Terpstra trend test indicated there were statistically significant decreases in the mean number of young produced per female in the 457 μg technical test substance/L treatment group when compared to the negative control (p ≤ 0.05).

G1 GROWTH
- Males: The mean total length and dry weight of male mysids in the negative control group was 8.06 mm and 0.82 mg, respectively. The mean total length of male mysids in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 8.16, 8.21, 8.04, 8.31, 8.11 and 7.97 mm, respectively. The mean dry weight of males in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 0.89, 0.83, 0.87, 0.84, 0.84 and 0.86 mg, respectively. Dunnett’s test and Jonckheere-Terpstra trend test indicated there were no statistically significant decreases in male mean total length and mean dry weight in any of the treatment groups, in comparison to the negative control (p > 0.05).

- Females: The mean total length and dry weight of female mysids in the negative control group was 8.03 mm and 1.02 mg, respectively. The mean total length of female mysids in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 8.32, 8.32, 8.07, 8.26, 8.17 and 8.08 mm, respectively. The mean dry weight of females in the 15, 28, 58, 113, 228 and 457 μg technical test substance/L treatment groups was 1.07, 1.05, 1.01, 1.09, 0.97 and 0.92 mg, respectively. Dunnett’s test and Jonckheere-Terpstra trend test indicated there were no statistically significant decreases in female mean total length in any of the treatment groups, in comparison to the negative control (p > 0.05). Dunnett’s test indicated there were no statistically significant decreases in mean dry weight in any of the treatment groups, in comparison to the negative control (p > 0.05). Consequently, the NOEC for growth (lengths and weights) was 457 μg technical test substance/L (equivalent to 153 μg cation/L), the highest concentration tested.

G2 SURVIVAL (96 HOURS)
Surviving mysids in the negative control group and in the treatment groups appeared normal with a few observations of lethargy and erratic swimming during the 96-hour evaluation period. These observations were consistent throughout the control and treatment groups. After 96-hours of exposure, survival of G2 juvenile mysids in the negative control and in the 15, 28, 58, 113 and 228 μg technical test substance/L treatment groups was 66.7, 98.1, 97.4, 100 and 73.7%, respectively. The 457 μg technical test substance/L treatment group did not produce the any of live young for evaluation. Fisher’s Exact test indicated there were statistically significant decreases in survival in the 228 μg technical test substance/L treatment group when compared to the negative control (p ≤ 0.05). Consequently, the NOEC for G2 juvenile survival was 113 μg technical test substance/L (equivalent to 38 μg cation/L). The LOEC for G2 juvenile survival was 228 μg technical test substance technical/L (equivalent to 76 μg cation/L).

Reported statistics and error estimates:

A detailed description of the statistics is presented in 'Any other information on materials and methods incl. tables'.

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

Test substance Mean Measured Concentration (µg/L)	Test substance Cation Equivalent Concentration (µg/L)	Juvenile Survival to Pairing on Day 14			Adult Survival to Test Termination on Day 28
		Number Originally Exposed	Number Surviving	Percent Survival	Number Alive at Pairing(1)	Number Surviving	Percent Survival
Negative Control	Negative Control	60	58	96.7	53	42	79.2
15	5.0	60	55	91.7	37	34	91.9
28	9.4	60	55	91.7	42	34	81.0
58	19	60	60	100	49	37	75.5
113	38	60	58	96.7	39	29	74.4
228	76	60	54	90.0	43	21	48.8*
457	153	60	36	60.0*	28	9	32.1*

* 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 14 due to the fact that extra females that cannot be used to form pairs and any immature mysids are discarded at the time of pairing on Day 14.

 

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

Test substance Mean Measured Concentration (µg/L)	Test substance Cation Equivalent Concentration (µg/L)	Mean Number of Young Produced Per Reproductive Day ± SD	Percent of Surviving Females Producing Young(1)	Mean Number of Young Per Surviving Female ± SD(1)
Negative Control	Negative Control	0.352 ± 0.092	100	5.5 ± 1.65
15	5.0	0.346 ± 0.249	76.5	4.9 ± 3.49
28	9.4	0.374 ± 0.189	76.5	5.4 ± 2.50
58	19	0.285 ± 0.086	94.4	4.0 ± 1.31
113	38	0.479 ± 0.171	78.6	6.8 ± 2.39
228	76	0.170 ± 0.069	64.3**	2.5 ± 1.29
457	153	0.000 ± 0.000*	0.0**	0.0 ± 0.00*

* Statistically significant decrease in reproduction and mean number of young per surviving female in comparison to the negative control using Dunnett’s test (p ≤ 0.05). Also, the decrease was significant according to the Jonckheere-Terpstra trend test (p ≤ 0.05).

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

(1) 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: Summary of Growth of Saltwater Mysids Exposed to the test substance

Test substance Mean Measured Concentration (µg/L)	Test substance Cation Equivalent Concentration (µg/L)	Mean Total Length ± SD (mm)		Mean Dry Weight ± SD (mg)
		Males	Females	Males	Females
Negative Control	Negative Control	8.06 ± 0.139	8.03 ± 0.157	0.82 ± 0.039	1.02 ± 0.062
15	5.0	8.16 ± 0.055	8.32 ± 0.162	0.89 ± 0.120	1.07 ± 0.131
28	9.4	8.21 ± 0.213	8.32 ± 0.203	0.83 ± 0.076	1.05 ± 0.028
58	19	8.04 ± 0.178	8.07 ± 0.262	0.87 ± 0.047	1.01 ± 0.077
113	38	8.31 ± 0.148	8.26 ± 0.052	0.84 ± 0.114	1.09 ± 0.041
228	76	8.11 ± 0.217	8.17 ± 0.129	0.84 ± 0.053	0.97 ± 0.039
457	153	7972	8.08 ± 0.363	0.862	0.92 ± 0.109

1) There were no statistically significant decreases in comparison to the negative control using Dunnett’s test (p > 0.05). Also, there were no decreases that were significant according to the Jonckheere-Terpstra trend test (p > 0.05) for male and female total length and male dry weight. The female dry weight data was non-monotonic and, therefore, Jonckheere-Terpstra trend test was not performed for this endpoint.

2) There was only one replicate present for growth measurements at termination. Therefore, standard deviation could not be calculated.

 

Table: Summary of Survival of G2 Saltwater Mysids Exposed to the test substance

Test substance Mean Measured Concentration (µg/L)	Test substance Cation Equivalent Concentration (µg/L)	Number Originally Exposed	Number Surviving	Percent Survival
Negative Control	Negative Control	49	48	98.0
15	5.0	3	2	66.7
28	9.4	53	52	98.1
58	19	39	38	97.4
113	38	53	53	100
228	76	19	14	73.7*
457	153	--(2)	--(2)	--(2)

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

1) The number of mysids originally exposed represents the sum of all G2 mysids initiated in each compartment within the entire treatment or control group.

2) The 457 μg/L did not produce any live young for evaluation.

Calculation of key result

The doses of the test substance were expressed in mg technical test material/L, which relates to an aqueous solution of the registered substance. The key effect levels are calculated by correction for the amount of water: 0.463 x 113 µg technical test material/L = 52.3 µg pure test substance/L. The recalculated LOEC value is 105.6 µg pure test substance/L

ADDITIONAL EFFECT VALUES

Juvenile survival (Day 0 - 14):

14-d NOEC: 228 μg technical test substance/L

14-d LOEC: 457 μg technical test substance/L

 

Adult survival (Day 14 - 28):

14-d NOEC: 113 μg technical test substance/L

14-d LOEC: 228 μg technical test substance/L

 

Overall G1 mortality:

7-d LC50: >457 μg technical test substance/L

14-d LC50: >457 μg technical test substance/L

21-d LC50: 457 μg technical test substance/L

28-d LC50: 235 μg technical test substance/L

 

Reproduction:

28-d NOEC: 113 μg technical test substance/L

28-d LOEC: 228 μg technical test substance/L

28-d MATC: 161 μg technical test substance/L

 

Growth (length and weight):

28-d NOEC: 457 μg technical test substance/L

 

G2 mortality:

96-h NOEC: 113 μg technical test substance/L

96-h LOEC: 228 μg technical test substance/L

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl, tables'.

Conclusions:

The 28-d NOEC of the technical test substance was determined to be 113 μg/L. The recalculated 28-d NOEC value for the pure substance was 52.3 μg/L.

Executive summary:

The long-term toxicity to aquatic (saltwater) invertebrates determined in a study according to OPPTS 850.1350 and in compliance with GLP criteria. In this study, groups of 60 (15 per replicate) juvenile mysid shrimp (G1; Americamysis bahia) were exposed for 28 days under flow-through conditions to nominal concentrations of 0 (control), 13, 25, 50, 100, 200 and 400 μg technical test substance/L. Test concentrations were analytically verified and determined to be <LOQ (control), 15, 28, 58, 113, 228 and 457 μg technical test substance/L (geometric mean). On Day 14 of the test, after 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. Overall, mysids were evaluated for survival, reproduction and growth. Offspring produced by the G1 mysids (the second generation, G2) were exposed to the same nominal test substance concentrations for approximately 96-hours following their release from the brood pouch. G2 mysids were evaluated for survival. Survival and reproduction in the G1 mysids were the most sensitive biological endpoints measured. There were statistically significant decreases in G1 mysid adult survival and percent of reproducing females in the 228 and 457 μg technical test substance/L treatment groups. The 28-day LC50 value was determined to be 235 μg technical test substance/L (equivalent to 79 μg cation/L), with a 95% confidence interval of 158 to 442 μg technical test substance/L (equivalent to 53 to 148 μg cation/L). Consequently, the NOEC, based on G1 mysid survival and reproduction, was 113 μg technical test substance/L (equivalent to 38 μg cation/L). The LOEC was 228 μg technical test substance/L (equivalent to 76 μg cation/L) and the MATC was calculated to be 161 μg technical test substance/L (equivalent to 54 μg cation/L). The recalculated 28-d NOEC and LOEC values for the pure substance were 52.3 and 105.6 μg/L, respectively.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

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:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

1996 (draft version)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests.

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Duplicate water samples were collected from each treatment and control group one day prior to the start of the test after conditioning the diluter for approximately five days. Duplicate water samples also were collected from test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. Stock solution samples were also collected to measure concentrations of the test substance. The samples were collected from mid-depth, placed in plastic vials, and three drops of 10% H3PO4 was added to each vial. One set of samples was processed immediately for analysis and the other set was stored under refrigeration for possible future analysis.

Vehicle:

no

Details on test solutions:

Individual stock solutions were prepared six times for each of the five concentrations tested. A primary stock solution was prepared by mixing a calculated amount of test substance into reverse osmosis water (2.5 grams into 250 mL final volume) at a nominal concentration of 10 mg paraquat dichloride technical/mL. Four secondary stock solutions were prepared in R.O. water at nominal concentrations of 0.63, 1.3, 2.5 and 5.0 mg paraquat dichloride technical/mL by proportional dilution of the primary stock (see table below). The stock solutions were mixed by inversion, and ranged in appearance from clear and light brown to clear and brown. Stock solutions were stored ambient in glass amber bottles, and aliquots of each stock were placed in the syringe pump every one to four days during the study.
The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 10.00 μL/minute where they were mixed with dilution water delivered at a rate of 100 mL/minute to achieve the desired test concentrations. The negative control received dilution water only.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by Wildlife International, Easton, Maryland.
Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.8 to 20.5ºC, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.7, measured with a Thermo Orion Model 525Aplus pH meter. Dissolved oxygen concentrations were ≥7.8 mg/L (≥87% of saturation), measured with a Thermo Orion Model 850Aplus dissolved oxygen meter.
During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. Daphnids were fed three times per day through Day 6 of the test and then were fed four times per day until the last day of the test. At each feeding, each test chamber was fed 0.75 mL of YCT, 1.5 mL of algae (Pseudokirchneriella subcapitata) and 0.50 mL of vitamin solution. This amount of feed is equal to approximately 0.71 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates.
The three adult daphnids used to supply neonates for the test were held for at least 12 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. To initiate the exposure, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 5 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the exposure. All transfers were made below the water surface using wide-bore pipettes.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Negative control: 137 ± 4 mg/L as CaCO3
Highest test concentration: 139 ± 4 mg/L as CaCO3

Test temperature:

20 ± 1 °C

pH:

8.1 to 8.3

Dissolved oxygen:

≥94% of saturation

Salinity:

Negative control: 172 ± 2 mg/L as CaCO3
Highest test concentration: 174 ± 2 mg/L as CaCO3

Conductivity:

Negative control: 366 ± 7 µS/cm
Highest test concentration: 374 ± 11 µS/cm

Nominal and measured concentrations:

Nominal: negative Control (0.0), 0.063, 0.12, 0.25, 0.50 and 1.0 mg test item /L
Mean measured concentrations of the test item contained in samples of test item collected at 0, 7, 14 and 21 days were 0.071, 0.14, 0.29, 0.59 and 1.2 mg test item/L ranging from 108 to 120% of nominal concentrations

Details on test conditions:

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 (Harvard Apparatus, South Natick, Massachusetts) 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 and was adjusted to provide approximately seven volume additions of test water in each test chamber per day. After mixing, the test solution in each mixing chamber was pumped into the appropriate replicate test chamber using a peristaltic pump (Cole-Parmer Instrument Company, Chicago, Illinois).
The syringe pumps used to deliver stock solutions to the mixing chambers were calibrated prior to the test. The pumps used to deliver the test solutions and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and calibrated/verified approximately weekly during the test. The proportion of the test solution that was pumped 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 ± 5% of the mean flow rate for the replicates. Delivery of test solutions to the test chambers was initiated six days 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 at least two times per day during the test.
The delivery system and the test chambers were placed in a temperature-controlled environmental chamber to maintain the target water temperature throughout the test period. Test chambers were 12-L glass aquaria filled with approximately 10 L of test water. The volume in the test chambers was maintained by an overflow port on one end of each test chamber. The daphnids were held in two test compartments suspended in each of the two test chambers per treatment. Test compartments were 300 mL glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens (220 μm) covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative chamber was 14 cm, while the depth of water in a representative test compartment was approximately 8 cm. All test chambers were labeled with the project number, test concentration and replicate designation.
Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber (Negative Control replicate A) at the beginning of the test using a SPER Scientific Model 840006 light meter.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: test substance

Basis for effect:

reproduction

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:

EC10

Effect conc.:

0.67 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: original value presented in the study (technical test item)

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: pure test substance

Basis for effect:

immobilisation

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.:

0.29 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: original value presented in the study (technical test item)

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

immobilisation

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:

EC10

Effect conc.:

0.62 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: original value presented in the study (technical test item)

Details on results:

Study results are based on mean measured concentrations.
Based on the mean measured concentrations and growth as the most sensitive indication of toxicity: No-Observed-Effect Concentration (NOEC) is 0.29 mg technical test material/L, respectively 0.097 mg test substance cation/L. The Low-Observed-Effect Concentration (LOEC) is determined at 0.59 mg technical test material/L, respectively at 0.20 mg test substance cation/L. The Maximum Acceptable Toxicant Concentration (MATC) is 0.41 mg technical test material/L and 0.14 mg test substance cation/L.

There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤0.29 mg technical test material/L. Growth, measured as length, was the most sensitive biological endpoint measured in this study. The effect on daphnid length was corroborated by a suppression in dry weight, though not statistically significant. Daphnids exposed to concentrations of ≥0.59 mg technical test material/L had statistically significant reductions in length in comparison to the negative control. Consequently, the NOEC, based on growth, was 0.29 mg technical test material/L (equivalent to 0.097 mg test substance cation/L). The LOEC was 0.59 mg technical test material/L (equivalent to 0.20 mg test substance cation/L) and the MATC was calculated to be 0.41 mg technical test material/L (equivalent to 0.14 mg test substance cation/L). The 21-day EC10 value for adult immobility was 0.62 mg technical test material/L (equivalent to 0.21 mg test substance cation/L) with a 95% confidence interval of 0.60 to 0.64 mg technical test material/L (equivalent to 0.20 to 0.21 mg test substance cation/L). The 21- day EC20 value for adult immobility was 0.68 mg technical test material/L (equivalent to 0.23 mg test substance cation/L) with a 95% confidence interval of 0.67 to 70 mg technical test material/L (equivalent to 0.22 to 0.23 mg test substance cation/L). The 21-day EC50 value for adult immobility was 0.80 mg technical test material/L (equivalent to 0.27 mg test substance cation/L) with a 95% confidence interval of 0.59 to 1.2 mg technical test material/L (equivalent to 0.20 to 0.40 mg test substance cation/L). The 21-day EC10 value for reproduction was 0.67 mg technical test material/L (equivalent to 0.22 mg test substance cation/L) with a 95% confidence interval of 0.63 to 0.69 mg technical test material/L (equivalent to 0.21 to 0.23 mg test substance cation/L). The 21-day EC20 value for reproduction was 0.76 mg technical test material/L (equivalent to 0.25 mg test substance cation/L) with a 95% confidence interval of 0.71 to 0.78 mg technical test material/L (equivalent to 0.24 to 0.26 mg test substance cation/L). The 21-day EC50 value for reproduction was 1.0 mg technical test material/L (equivalent to 0.34 mg test substance cation/L) with a 95% confidence interval of 0.97 to 1.1 mg technical test material/L (equivalent to 0.32 to 0.37 mg test substance cation/L).

 

Effect values for endpoints, based on mean measured concentrations after 21 days of exposure

		TEchnical test material (mg/L)		test substance cation (mg/L)
Endpoint and effect		Value	95% CI	Value	95% CI
EC10	(Immobility)	0.62	0.6 -0.64	0.21	0.2-0.21
EC20	(Immobility)	0.68	0.67-0.7	0.23	0.22-0.23
EC50	(Immobility)	0.8	0.59-1.2	0.27	0.2-0.4
EC10	(Reproduction)	0.67	0.63-0.69	0.22	0.21-0.23
EC20	(Reproduction)	0.76	0.71-0.78	0.25	0.24-0.26
EC50	(Reproduction)	1	0.97-1.1	0.34	0.32-0.37

 

Calculation of key result

The doses of the test substance were expressed in mg technical test material/L, which relates to an aqueous solution of the registered substance (46.3% wt/wt purity). The key effect levels are calculated by correction for the amount of water: 0.463 x technical test material/L = pure test substance/L. The key recalculated values are presented in the 'Effect concentrations' field.

Validity criteria fulfilled:

yes

Conclusions:

The 21-d NOEC of the technical test material was determined to be 0.29 mg/L based on immobilisation. The recalculated 21-d NOEC value for the pure test substance was 0.13 mg/L.

Executive summary:

The test substance was tested using a 21 days exposure study, according to the OECD guideline 211. Cladoceran (Daphnia magna) was used as the test organism for the study. The test organism age was <24 hours old at initiation, as Neonates. During the exposure the temperature was estimated within 20 ± 1ºC, the dissolved oxygen was estimated to be ≥94% of saturation and the pH between 8.1 and 8.3. The test item was measured at nominal concentrations of: negative Control (0.0), 0.063, 0.12, 0.25, 0.50 and 1.0 mg test item /L. Mean measured concentrations of the test item contained in samples of test item collected at 0, 7, 14 and 21 days were 0.071, 0.14, 0.29, 0.59 and 1.2 mg test item/L ranging from 108 to 120% of nominal concentrations, and indicated that test solutions had been properly dosed.

There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤0.29 mg technical test material/L. Growth, measured as length, was the most sensitive biological endpoint measured in this study. The effect on daphnid length was corroborated by a suppression in dry weight, though not statistically significant. Daphnids exposed to concentrations of ≥0.59 mg technical test material/L had statistically significant reductions in length in comparison to the negative control. Consequently, the NOEC, based on growth, was 0.29 mg technical test material/L (equivalent to 0.097 mg test substance cation/L). The LOEC was 0.59 mg technical test material/L (equivalent to 0.20 mg test substance cation/L) and the MATC was calculated to be 0.41 mg technical test material/L (equivalent to 0.14 mg test substance cation/L). The 21-day EC10 value for adult immobility was 0.62 mg technical test material/L (equivalent to 0.21 mg test substance cation/L) with a 95% confidence interval of 0.60 to 0.64 mg technical test material/L (equivalent to 0.20 to 0.21 mg test substance cation/L). The 21- day EC20 value for adult immobility was 0.68 mg technical test material/L (equivalent to 0.23 mg test substance cation/L) with a 95% confidence interval of 0.67 to 70 mg technical test material/L (equivalent to 0.22 to 0.23 mg test substance cation/L). The 21-day EC50 value for adult immobility was 0.80 mg technical test material/L (equivalent to 0.27 mg test substance cation/L) with a 95% confidence interval of 0.59 to 1.2 mg technical test material/L (equivalent to 0.20 to 0.40 mg test substance cation/L). The 21-day EC10 value for reproduction was 0.67 mg technical test material/L (equivalent to 0.22 mg test substance cation/L) with a 95% confidence interval of 0.63 to 0.69 mg technical test material/L (equivalent to 0.21 to 0.23 mg test substance cation/L). The 21-day EC20 value for reproduction was 0.76 mg technical test material/L (equivalent to 0.25 mg test substance cation/L) with a 95% confidence interval of 0.71 to 0.78 mg technical test material/L (equivalent to 0.24 to 0.26 mg test substance cation/L). The 21-day EC50 value for reproduction was 1.0 mg technical test material/L (equivalent to 0.34 mg test substance cation/L) with a 95% confidence interval of 0.97 to 1.1 mg technical test material/L (equivalent to 0.32 to 0.37 mg test substance cation/L).

The recalculated 21-d NOEC value for the pure test substance was 0.13 mg/L for immobilisation.

Description of key information

The 21-d NOEC value is 0.13 mg pure substance/L (recalculated) for freshwater invertebrates (Daphnia magna), OECD 211, Claude 2014

The 28-d NOEC value is 52.3 µg pure substance/L (recalculated) for the saltwater mysid (A. bahia), OPPTS 850.1350, Claude, 2014

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.13 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

52.3 µg/L

Additional information

Table: Overview of aquatic invertebrate long-term toxicity data

Species	Guideline/ GLP	Endpoint	Effect value	Comment	Reference
Daphnia magna (freshwater)	OECD 211/ GLP	21-d NOEC	0.13 mg/L (recalculated)	Flow-through regime. The mean measured concentrations were used for the effect values	Claude 2014
Americamysis bahia (saltwater)	OPPTS 850.1350/ GLP	28-d NOEC	52.3 µg/L (recalculated)	Flow-through regime. The mean measured concentrations were used for the effect values	Claude 2014