Diquat dibromide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP study

Qualifier:

according to guideline

Guideline:

other: EPA-660/3-75-009

Version / remarks:

1975

Deviations:

not specified

Principles of method if other than guideline:

Besides the EPA-660/3-75-009 guideline, the study was also based on a EPA approved protocol called 'Protocol for Daphnia LC50 Studies'.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

400 mL aliquot of the treated water was employed for 0 hour water analysis and toxicant quantitative analysis. Sampling for toxicant concentrations analysis was carried out after 48 hours exposure as well. Sample analyses were done after 48 hours employing the test vessels containing Daphnia and reported as milligrams test substance per liter of diluent water (mg/L).

Vehicle:

no

Details on test solutions:

Test substance concentration levels were prepared by directly adding 100 mg of the test substance into 1000 mL stock of test water, followed by vigorous mixing in a blender for 1 minute. This stock solution was used to prepare serial dilutions of 1.01, 1.68, 2.8 and 4.66 mg test substance cation/L. Each liter of treated water was divided into several aliquots: three 200 mL aliquots were each delivered into 250 mL glass beakers which served as test vessels for the daphnia. The rest aliquots are for water analysis.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Cultured in the test facility
- Culture condition: 17 ± 1.5°C in glass tanks
- Feeding during cultivation: Feed on live yeast
- Age at study initiation: < 24 hours old
- Stage and instar at study initiation: 1st stage nymphs
- Feeding during test: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

40 - 42 mg/L as CaCO3

Test temperature:

17 ± 1.5 °C

pH:

7.9 - 8.1

Dissolved oxygen:

92 - 95% saturation

Nominal and measured concentrations:

- Nominal concentration: 0 (control), 1.01, 1.68, 2.80 and 4.66 mg test substance cation/L
- Measured concentration (at 0 hour): 0 (control), 0.94, 1.65, 2.28 and 3.92 mg test substance cation/L, respectively.
- Measured concentration (at 48 hours): 0 (control), 0.84, 1.37, 2.24 and 3.83 mg test substance cation/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel:
250 mL glass beakers
- Volume of solution:
200 mL
- No. of organisms per vessel:
10
- No. of vessels per concentration: 3
- No. of vessels per control:
3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Deionized municipal water reconstituted to a hardness of 40 mg CaCO3/L was stored at 17 ± 1.5°C in glass and polyethylene tanks and conditioned by intense aeration for a period of two weeks prior
to use as culture water and/or diluent test water. During the test period, water quality was analysed
- Culture medium different from test medium:
No
- Intervals of water quality measurement:
Temperatures were continuously monitored and a thermistor probe placed in the geometric center of a control vessel containing untreated water.

OTHER TEST CONDITIONS
- Photoperiod:
8 hours darkness and 16 hours of light with a 45 minute, 3 phase, light intensity transition period at the beginning and end

EFFECT PARAMETERS MEASURED
Daphnia mortality was read after 24 hours and 48 hours exposure to treatments. Death was defined by organisms being immobile or by lack of movement except for minor activity of appendages.

RANGE-FINDING STUDY
Preliminary range finding tests were performed to define a narrower range of nominal test substance concentrations that would yield about 35 - 65% mortality in a subsequent definitive test (2.2 - 10 mg of the test substance/L).

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.22 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: pure test substance

Basis for effect:

mortality

Remarks on result:

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

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

1.19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: test substance cation

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

2.519 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: test substance cation

Basis for effect:

mortality

Remarks on result:

other: original value presented in study

Details on results:

An overview of the result is provided in Table 1 in 'Any other informatoion on results incl. tables'.
All test organisms survived in the control groups after 24 and 48 hours exposure. The mortality at 24 hours was 0, 13%, 40% and 83% in 1.01, 1.68, 2.80 and 4.66 mg cation/L (nominal concentration), respectively. The mortality at 48 hours ranged from 33.3% at the 1.01 mg test substance cation/L to 100% at the 2.8 mg cation/L.

Reported statistics and error estimates:

Chi square statistical analysis was done to give a median lethal concentration (LC50) after 24 and 48 hours exposure (based on 0 hour toxicant analytical data), both at the 95% confidence limits.

Table 1. percent mortality of the test organisms after 24 and 48 hours exposure to the test material

0 hour			24 hours				48 hours
Nominal concentration (mg/L)		Measured concentration (mg test substance cation/L)	Percent Mortality				Measured concentration (mg test substance cation/L)	Percent Mortality
			Replicate			Mean		Replicate			Mean
			A	B	C			A	B	C
A1	B2
10	4.66	3.92	60	100	90	83	3.83	100	100	100	100
6	2.8	2.28	50	20	50	40	2.24	100	100	100	100
3.6	1.68	1.65	20	10	10	13	1.37	80	70	60	70
2.2	1.01	0.94	0	0	0	0	0.84	50	30	20	33.3
Control		0	0	0	0	0	0	0	0	0	0
LC50			2.5195				1.1898
Upper 95% confidence limit			3.1031				1.4408
Lower 95% confidence limit			2.0456				0.9825

Note. The LC50 was calculated based on analytical concentration of the cation part of the test material at 0 hour.

1. The test material

2. Cation

 

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 registered substance divided by 184.2 g/mol molecular weight of cation species).:

1.868 x 1.1898 mg/L = 2.22 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour LC50 was determined to be 1.1898 mg test substance cation/L (95% confidence limit of 0.98 to 1.44 mg/L), equivalent to 2.22 mg pure test substance/L (95% confidence limit of 1.83 to 2.69 mg/L).

Executive summary:

To determine the 48-hour acute toxicity of the test substance to the water flea (Daphnia magna), the exposure was conducted under static conditions following EPA 660/3-75-009 guideline. The test was not in compliance with GLP criteria. Less than 24 hours old Daphnia magna were exposed to the test substance at measured concentrations of 0.94, 1.65, 2.28 and 3.92 mg/L, respectively, for 48 hours. Three groups of ten individuals were exposed at each test concentration, at a temperature of 17°C. Dissolved oxygen, water hardness and pH were determined at the beginning and end of the study and temperature was monitored continuously and found to be in an acceptable range. Mortalities were recorded at 24 and 48 hours. All test organisms survived in the control groups after 24 and 48 hours exposure. The mortality at 24 hours was 0, 13%, 40% and 83% at 0.94, 1.65, 2.28 and 3.92 mg test substance cation/L, respectively. The mortality at 48 hours ranged from 33.3% at 1.01 mg/L to 100% at 2.8 mg/L. Based on the observed mortality, the 24-hour LC50 was calculated as 2.52 mg test substance cation/L and the 48 hour LC50 was 1.19 mg test substance cation/L, equivalent to 4.71 and 2.22 mg pure test substance/L, respectively.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jun to 19 Jun 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-3 (Estuarine/Marine Fish, Mollusk, or Shrimp Acute Toxicity Test)

Version / remarks:

October 1982

Deviations:

yes

Remarks:

See protocol deviation in 'Any other information on materials and methods incl. tables'

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were removed from each treatment level and control at 0, 48 and 96 hours. Samples for 0-hour analyses were removed from each treatment level prior to the time in which replicate solutions were divided. The 48 and 96-hour samples were from alternating replicate solutions.

Vehicle:

no

Details on test solutions:

A clear, colorless stock solution of 1.0 mg/mL was prepared by diluting 0.10 g of the test material with distilled water to volume in a 100 mL volumetric flask. The test solutions were prepared by adding the appropriate volumes of the stock solution and dilution water to each test vessel to total 2500 mL. Each solution was mixed with a magnetic stirrer and teflon-coated stir bar. Duplicate 1000 mL aliquots were removed and served as exposure solutions. For the control solution, the same dilution water was used but with no test material in it.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid shrimp
- Age at study initiation: ≤ 24 hours old

CULTIVATION
- Photoperiod: 16 hours of light and 8 hours darkness
- Light intensity and location: 70 - 140 footcandles at the culture solution surface
- Feeding during cultivation: Feed with live brine shrimp nauplii twice daily and the soil nematode (panagrellus redivivus) three times weekly.
- Feeding during test: Mysids were fed brine shrimp nauplii each day of exposure
- Temperature: 25 ± 1 °C (maintained by commercial aquarium heater)

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25 ± 1°C

pH:

8.0

Dissolved oxygen:

5.9 - 6.8 mg O2/L

Salinity:

30‰

Nominal and measured concentrations:

- Nominal concentration: 0.78, 1.3, 2.2, 3.6 and 6.0 mg test material/L
- Measured conentration: 0.81, 1.3, 2.3, 3.5 and 5.7 mg test material/L, respectively. (See Table 1 in 'Any other information on materials and methods incl. tables')

Details on test conditions:

TEST SYSTEM
- Volume of solution: 1000 mL (depth was 3.5 cm with a surface area of 280 cm2)
- Type: Closed (covered with plate glass to minimise evaporation)
- Aeration: No
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture and dilution water were prepared by filtering natural seawater collected from the Care Cod Canal, Bourne, Massachusetts, which derives water from either Cape Cod Bay or Buzzards Bay depending upon tidal direction. The seawater was filtered through a 5-micrometer porosity polypropylene core filter and an activated carbon bed prior to use. The dilution water was characterized as having a salinity of 30‰ and a pH of 8.0. This water was aerated prier to use to ensure the dissolved oxygen concentration was greater than 90% of saturation.
- Intervals of water quality measurement: The pH, dissolved oxygen concentration, temperature and salinity were measured at 0 hour and each subsequent 24-hour exposure interval in all replicate treatment and control solutions.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours darkness (same as during cultivation)
- Light intensity and location: 50 - 80 footcandles at the surface of the solutions

EFFECT PARAMETERS MEASURED
Biological observations and observations of the physical characteristies of each replicate test solution were made and recorded at 0 , 24 , 48 , 72 and 96 hours of exposure. The number of dead mysid shrimp in each replicate test solution was recorded at 24 , 48 , 72 and 96 hours. Dead mysids were removed daily.

RANGE-FINDING STUDY
- Test concentrations: 1.3 - 10 mg/L
- Results used to determine the conditions for the definitive study: Results of the preliminary exposure of mysids to the test substance indicated the LC50 would be approximately 3.0 mg/L (nominal concentration). Analyses of the preliminary exposure solutions (low and high treatment levels) resulted in measured concentrations ranging from 85 to 120% of the nominal levels. The nominal concentrations selected for the definitive exposure were 0.78, 1.3, 2.2, 3.6 and 6.0 mg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: pure test substance

Basis for effect:

mortality

Remarks on result:

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

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

liquid concentrate

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Remarks:

95% C.L.: 1.7 - 2.3 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

liquid concentrate

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Remarks:

95% C.L.: 2.3 - 3.2 mg/L

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

4.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

liquid concentrate

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Remarks:

95% C.L.: 3.4 - 5.5 mg/L

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 5.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Remarks:

liquid concentrate

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Details on results:

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

All mysids survived in the control and 0.81 mg/L treatment during the entire experimental period. After 24 hours exposure to the test substance, no dead organisms were observed in the 1.3 and 2.2 mg/L treatment groups, in 3.5 and 5.7 mg/L treatment groups mortality was 15% and 5%, respectively. At 48 hours 5, 20, 30 and 75% mortality showed in 1.3, 2.3, 3.5 and 5.7 mg/L treatment groups, respectively. Another 24 hours later mortality increased to 10, 40, 65 and 95% in those treatments. At the end of the test 25, 50, 95 and 100% mortality was found in 1.3, 2.3, 3.5 and 5.7 mg test material/L, respectively.

Reported statistics and error estimates:

Three statistical methods were available in the computer program: moving average angle analysis, probit analysis, and nonlinear interpolation with 95 % confidence intervals calculated by binomial probability. Moving average angle and probit analyses yield statistically sound results only if at least two concentrations produce a mortality of between 0 and 100% of the test organism population. The selection of reported LC50 values and 95% confidence intervals was based upon an examination of the data base and the results of the computer analysis. Selection criteria included the establishment of a concentration-effect relationship (mortality), the number of concentrations causing partial responses, and the span of responses bracketing the LC50 value. If two or more statistical methods produced acceptable results, then the method which yielded the smallest 95% confidence interval was selected.

Table 2. Concentrations tested, corresponding mortalities and observations made during the 96-hour static exposure of mysid shrimp (Mysidopsis bahia) to the test material.

Nominal concentration	Mean measured concentration	Mortality (%)
		24 hours			48 hours			72 hours			96 hours
		A	B	mean	A	B	mean	A	B	mean	A	B	mean
Control	Control	0	0	0	0	0	0	0	0	0	0	0	0
0.78	0.81	0	0	0	0	0	0	0	0	0	0	0	0
1.3	1.3	0	0	0	0	10	5	0	20	10^b	20	30	25
2.2	2.3	0	0	0^a	20	20	20^a	50	30	40	60	40	50
3.6	3.5	10	20	15^b	20	40	30	50	80	65^b	100	90	95
6.0	5.7	10	0	5^a	70	80	75^c	90	100	95^b	100	100	100

a. Several surviving mysids were lethargic.

b. One mysid was lethargic.

c. All of the surviving mysids were lethargic.

 

Calculation of key result

The original effect levels were expressed as test material, which is a liquid concentrate (41.4% purity) of the test substance. The key effect levels are re-calculated to correct for the amount of water:

41.4% x 2.0 mg/L = 0.83 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 value for mysid shrimp exposed to the test material was calculated by probit analysis to be 2.0 mg/L, with 95% confidence intervals of 1.7 - 2.3 mg/L. This LC50 value is equivalent to 0.83 mg pure test substance/L.

Executive summary:

To determine the 96-hour acute toxicity of the test substance to mysid shrimp (Mysidopsis bahia), the exposure was conducted under static conditions following EPA 72-3 guideline. The test was in compliance with GLP criteria. Less than 24 hours old mysids were exposed to measured concentrations of 0.81, 1.3, 2.3, 3.5 and 5.7 mg test material/L and analyzed by spectrophotometry. The study was carried out in 30‰ natural seawater, at pH 8.0 and 25 ± 1°C. Duplicate test vessels were used for each of the exposure concentration and control. The mortality of the test organisms was studied in 24 hours interval.

All mysids survived in the control and 0.81 mg/L treatment during the entire experimental period. At the end of the test, 25, 50, 95 and 100% mortality was found at 1.3, 2.3, 3.5 and 5.7 mg/L, respectively. The 96-hour LC50 value for mysid shrimp exposed to the test substance were calculated by probit analysis to be 2.0 mg/L, with 95% confidence intervals of 1.7 - 2.3 mg/L. This LC50 value of the test substance is equivalent to 0.83 mg pure test substance/L. The slope of the concentration-response (mortality) curve, as calculated by probit analysis was 5.4.The NOEC was determined to be 0.81 mg/L of the test material.

Description of key information

Freshwater, 48-h LC50 = 2.22 mg test substance/L, Daphnia magna, EPA 660/3-75-009, Wheeler 1978

Marine water, 96-h LC50 = 0.83 mg test substance/L, Americamysis bahia (previously Mysidopsis bahia), EPA 72-3, Hoberg 1987

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

2.22 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

LC50

Effect concentration:

0.79 mg/L

Additional information

Freshwater

To determine the 48-hour acute toxicity of the test substance to the water flea (Daphnia magna), the exposure was conducted under static conditions following EPA 660/3-75-009 guideline. The test was not in compliance with GLP criteria. Less than 24 hours old Daphnia magna were exposed to the test substance at measured concentrations of 0.94, 1.65, 2.28 and 3.92 mg/L, respectively, for 48 hours. Three groups of ten individuals were exposed at each test concentration, at a temperature of 17°C. Dissolved oxygen, water hardness and pH were determined at the beginning and end of the study and temperature was monitored continuously and found to be in an acceptable range. Mortalities were recorded at 24 and 48 hours. All test organisms survived in the control groups after 24 and 48 hours exposure. The mortality at 24 hours was 0, 13%, 40% and 83% at 0.94, 1.65, 2.28 and 3.92 mg test substance cation/L, respectively. The mortality at 48 hours ranged from 33.3% at 1.01 mg/L to 100% at 2.8 mg/L. Based on the observed mortalitythe 48 hour LC50 was 1.19 mg test substance cation/L, equivalent 2.22 mg pure test substance/L.

 

Marine water

To determine the 96-hour acute toxicity of the test substance to mysid shrimp (Mysidopsis bahia), the exposure was conducted under static conditions following EPA 72-3 guideline. The test was in compliance with GLP criteria. Less than 24 hours old mysids were exposed to measured concentrations of 0.81, 1.3, 2.3, 3.5 and 5.7 mg test material/L and analyzed by spectrophotometry. The study was carried out in 30‰ natural seawater, at pH 8.0 and 25 ± 1°C. Duplicate test vessels were used for each of the exposure concentration and control. The mortality of the test organisms was studied in 24 hours interval.

All mysids survived in the control and 0.81 mg/L treatment during the entire experimental period. At the end of the test, 25, 50, 95 and 100% mortality was found at 1.3, 2.3, 3.5 and 5.7 mg/L, respectively. The 96-hour LC50 value for mysid shrimp exposed to the test substance were calculated by probit analysis to be 2.0 mg/L, with 95% confidence intervals of 1.7 - 2.3 mg/L. This LC50 value of the test substance is equivalent to 0.83 mg pure test substance/L. The slope of the concentration-response (mortality) curve, as calculated by probit analysis was 5.4.The NOEC was determined to be 0.81 mg/L of the test material.