Paraquat-dichloride

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 May 1998 to 8 Jun 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

ASTM E1383 (Sediment Toxicity Test (Media: Sediment-freshwater))

Version / remarks:

1993

Qualifier:

equivalent or similar to guideline

Guideline:

other: SETAC-Europe Guidance Document on Sediment Toxicity Tests and Bioassays for Freshwater and Marine Environments.

Version / remarks:

1993

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Overlying water: A 10 mL aliquot of overlying water was removed from approximately 5 cm below the water surface and analysed by liquid scintillation counting (LSC) to determine total radioactivity.
- Sediment: On day 0 after sampling the overlying water, the remaining overlying water was discarded. The sediment was then mixed, subsampled, air dried, and 5 x 0.2 g aliquots weighed into combustion cones and combusted to determine total radioactivity.

Vehicle:

no

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Method of mixing and details of spiking: Test systems (replicates A - E) were prepared by treating sediment-water slurries (200 g dry weight sediment : 500 mL water) in a 1 litre glass jars with the test chemical in test water to give a nominal concentration of 100 mg ion/kg dry weight sediment.
- Equilibration conditions: chambers were then placed in a water bath at 20 ± 2°C and allowed to stand undisturbed for three days to allow the sediment to settle
- Controls: Control systems (A - D) were prepared in the same way, except no test chemical was spiked into the systems

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: Non-biting midge
- Justification for species other than prescribed by test guideline: Chironomus riparius is a member of the widespread dipteran insect family Chironomidae, commonly referred to as non-biting midges. It has four sediment-dwelling, larval instars which are widely used in sediment toxicity testing
- Age of parental stock: Mixed age cultures were maintained.
- Breeding conditions: The cultures were maintained in hard water at approximately 20°C on a 16 hour: 8 hour light:dark cycle. Culture vessels were 5 litre plastic aquaria containing water approximately 12 cm deep overlying approximately 2 cm of silver sand. Cultures were fed ground Tetramin® (high protein fish food) as required.
- Handling of egg masses and larvae: To obtain organisms for the test, egg ropes were removed from the cultures and hatched in small plastic trays. On the day of hatching, the larvae were transferred to large glass crystallising dishes containing hard water overlying a thin layer of silver sand. Gentle aeration was provided and Chlorella vulgaris and ground Tetramin® were added as food.
- Age of animals at beginning of exposure: 2 days

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Duration:

21 d

Exposure phase:

total exposure duration

Hardness:

172 mg/L as CaCO3

Test temperature:

19.8 - 20.2 °C

pH:

8.4 - 8.7

Dissolved oxygen:

9.0 - 9.1 mg O2/L

Conductivity:

500 - 520 µS/cm

Nominal and measured concentrations:

- Nominal concentration: 100 mg/kg
- Measured concentration sediment: 94 mg/kg
- Measured concentration overlying water: 0.020 µg/L at t=0, 0.064 µg/L at t=7d, 0.121 µg/L at t=14d and 0.026 µg/L at t=21d.
Concentrations in the water phase as percentage of applied were low, ranging from 0.2% on day 0, before the organisms were introduced to 1.2% on day 14. Expressed as 14C-test substance equivalents, the concentration ranged from 0.021 to 0.121 µg/L. A previous study has established the stability of 14C-test substance in water-sediment systems and so the data show that virtually all the test substance in the system was adsorbed to the sediment. Indeed, it is likely that much of this 14C-residue in the water phase at days 7 and 14 was associated with suspended sediment, as on these days there was a significant amount of suspended material in the water phase. This suspended material is a consequence of the activity of the growing chironomid larvae.

Details on test conditions:

TEST SYSTEM
- Test container: The sediment/water test systems consisted of 3-litre tall form glass beakers containing 200 g (dry weight) treated sediment and 2 litres water. The test vessels were covered throughout the test to reduce evaporation.
- Introduction of individuals: First instar C. riparius were introduced into the treated and control systems after the test substance settled in the chambers. The day of introduction of the test organisms was designated day 0. The treated replicate E system was used only for day 0 physico-chemical measurements and then destructively sampled for analysis of water and sediment for test substance residues.
- Aeration: Yes; the test vessels were gently aerated

EXPOSURE REGIME
- No. of organisms per container: 25
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- Feeding regime: Ground Tetramin® (75 mg) was added to each test chamber after the addition of the organisms on day 0. Additional feeding was done on days 4, 8 and 11, when
100, 75 and 50 mg, respectively were added to each test chamber.

OVERLYING WATER CHARACTERISTCS
The dilution water used to prepare the test concentrations was a hard water (172 mg 1-1 CaCO3) produced by mixing dechlorinated mains water with the same water which had been deionised using a reverse osmosis system.
- Alkalinity: 96 mg/L as CaCO3

CHARACTERIZATION OF ARTIFICIALSEDIMENT
Artificial sediment was prepared at the testing facility in accordance with OECD guideline No. 207.
- % dry weight of sphagnum moss peat: 10 (air dried and finely ground prior to mixing) CaCO3 at 5 g kg
- Particle size distribution
- % fine silica sand: 70
- % koalin clay: 20

SYSTEM QUALITY MEASUREMENTS
- Dissolved oxygen and pH: Dissolved oxygen and pH were determined in one treated and control replicate on days 0 (prior to spiking) and 21, using YSI Model 57 and pHM250 meters respectively.
- Specific conductivity: Specific conductivity was determined in all test vessels on days 0 (prior to spiking) and 21, using a YSI Model 33 meter.
- Hardness and alkalinity: The hardness and alkalinity were measured in the test water used to prepare the test systems.
- Temperature: Temperature was monitored in the water bath by means of a temperature probe, with readings taken every 30 minutes.

OTHER TEST CONDITIONS
- Photoperiod: 16 hour light: 8 hour dark cycle (illumination from above)
- Light intensity: Approximately 750 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
From day 10 to day 21, the test systems were observed daily for emerged adults and the number of males and females were recorded. After each daily assessment, the midges were removed from the test systems and discarded. On day 21 the study was terminated.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

138.1 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

other: pure test substance

Basis for effect:

emergence rate

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.:

100 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

emergence rate

Remarks on result:

other: original value presented in the study (test substance ion)

Details on results:

The C. riparius emergence data are presented in 'any other information on results incl. tables'. There was no apparent difference in the pattern of emergence between control and treated systems. There was a single adult emerged on day 12 from one replicate of the treated vessels with day 13 being the day of first emergence from the other treated and the control replicates. The day of emergence for the last adult was day 18 for both control and treated, by which time cumulative total emergence was 97 and 99%, respectively with mean emergence times of 14.1 and 14.0 days.

Table: Number of emerged midges

Study Day	Treatment Male/Female
	Control				100 mg test substance/kg
	A	B	C	D	A	B	C	D
10	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
11	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
12	0/0	0/0	0/0	0/0	0/0	1/0	0/0	0/0
13	8/0	9/0	10/0	2/1	5/0	9/1	5/0	9/1
14	4/7	2/4	2/8	7/4	6/8	4/6	7/2	4/7
15	0/5	3/6	1/3	0/7	0/5	0/3	1/9	0/3
16	0/0	0/1	0/0	0/2	0/1	0/1	0/1	0/0
17	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
18	0/1	0/0	0/0	0/0	0/0	0/0	0/0	0/1
19	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
20	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
21	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
Total / Rep	25	25	24	23	24	25	25	25
Total %	97				99
Mean Emergence Time (days)	14	14.1	13.8	14.4	14	13.7	14.3	13.9
Overall Mean Emergence Time	14.1 days				14.0 days

          

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

NOEC: (100/72.4) x 100 mg test substance cation/kg = 138.1 mg pure test substance/kg.

Validity criteria fulfilled:

not specified

Conclusions:

The test substance applied to sediment at a concentration of 100 mg test substance ion/kg had no effect on the survival or development of Chironomus riparius. This effect value corresponds to a recalculated value of 138.1 mg pure substance/kg.

Executive summary:

The toxicity of the test substance to sediment-dwelling organisms was determined in a GLP-compliant study equivalent or similar to ASTM E1383 and SETAC recommendations on sediment testing. In this study, Chironomus riparius larvae (100 per treatment) were exposed to [14C]-labelled test substance in laboratory sediment-water systems at 20 ± 2°C using an artificial sediment. Test systems were prepared by applying to give a nominal concentration of 100 mg test substance ion/kg dry weight sediment in a sediment-water slurry system. Beginning at day 10, the test systems were observed daily for emerging adults, and the numbers of males and females were recorded. The test was terminated after 21 days. The concentration of the chemical on the sediment and in overlying water was determined at the time of introduction of the organisms. In addition water phase concentrations were determined on days 7, 14 and day 21. The measured radioactivity in the sediment on day 0 was equivalent to 94% of applied. Measured concentrations of radioactivity in the overlying water on days 0, 7, 14 and 21 were equivalent to 0.2 to 1.2% of applied. There was no apparent difference in the pattern of emergence between control and treated systems. Total emergence was 97 and 99%, respectively with mean emergence times of 14.1 and 14.0 days. Based on these findings, the 21-d NOEC was determined to be 100 mg test substance ion/kg, which corresponds to a recalculated value of 138.1 mg pure

substance/kg .

Reference 2

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 Nov 2014 to 21 Nov 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1735 (Whole Sediment Acute Toxicity of Invertebrates, freshwater)

Version / remarks:

1996

Deviations:

yes

Remarks:

see 'Any other information on materials and methods incl. tables'

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

See 'Details on analytical methods'

Vehicle:

no

Details on sediment and application:

RADIOLABELED STOCK SOLUTION PREPARATIONS
A primary radiolabeled stock solution was prepared by quantitatively transferring the entire amount of test substance to a Nalgene™ container and diluting it to 11 mL total volume with purified reagent water. Duplicate 10.0-μL aliquots of the stock solution were then transferred to glass vials containing 10 mL of Ultima Gold cocktail and mixed well prior to being assayed by liquid scintillation counting (LSC). Based on this analysis, the supplied specific activity was 202.3 μCi/mg (449,106 dpm/μg as test substance and 620,312 dpm/μg as test substance cation), the stock solution was determined to have a concentration of 0.960 mg/mL.

NONRADIOLABELED STOCK SOLUTION PREPARATION
A 25 mg test substance/mL primary nonradiolabeled stock solution was prepared by placing 0.6292 g (0.6254 g as active ingredient) of test substance into a volumetric flask and bringing it to a final volume of 25 mL with deionized water. The stock solution was observed to be light brown in color with no visible undissolved test substance following preparation. This primary nonradiolabeled stock solution was used to prepare the dosing solutions.

SEDIMENT DOSING STOCK SOLUTIONS
Dosing stock solutions for all test concentrations were prepared by adding appropriate volumes of both radiolabeled and nonradiolabeled primary stock solutions to a 10-mL volumetric stock. Dosing stock solutions for all test concentrations were then brought to a final volume of 10 mL with deionized water.

APPLICATION OF THE TEST SUBSTANCE TO SEDIMENT
A jar-rolling technique was used to apply the test substance to the sediment. The appropriate volume of each dosing stock solution was applied directly to 1.7 kg of wet sediment (0.7745 kg dry weight based on a percent solid value of 45.56%) in a 4-L glass jar. The test substance was then applied to the sediment. The jars were sealed and positioned horizontally on the rolling mill. Each jar was then rolled for four hours at room temperature at approximately 15 rpm. Following four hours of rolling, the jars were stored upright at 2 to 8 ºC. The dosed sediments were allowed to equilibrate for a 20-day period in the refrigerator. The length of the equilibration period used was based on partitioning data previously collected for the test compound diquat dibromide, applied to sediment. Because of the similar chemical characteristics of these two compounds, it was determined that a similar equilibration period of spiked sediments was appropriate. Once a week during the 20-day equilibration period and prior to addition into the replicate test vessels, the jars were mixed on the rolling mill for an additional two hours at room temperature to ensure the sediment was homogeneous.
A control sample was prepared in a similar manner as the treated sediment by adding 9.0 mL of deionized water, containing no test substance, to 1.7 kg of wet sediment and processed in a similar manner as the treated sediments.

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM
- Common name: freshwater amphipod
- Source: The amphipods used during this study were obtained from laboratory cultures maintained at the test facility.
- Feeding during test: Yes, see 'Details on test conditions'

HOLDING
- Procedure: Prior to exposure initiation, amphipods were maintained in 38-L glass aquaria containing approximately 31 L of culture water under flow-through conditions. The source of both the culture water and the overlying water used during the test was laboratory well water. Eight day old amphipods used in the test were collected from reproducing adult amphipods removed from the main culture tanks nine days prior to exposure initiation. The adult amphipods were placed in 9.5-L aquaria (isolation tanks) containing approximately 8 L of water. Neonate amphipods (≤ 24 hours old) produced by these isolated adults were then removed from the isolation tanks and pipetted into 1-L beakers containing approximately 0.80 L of laboratory well water. Each 1-L beaker was stocked with approximately 225 neonate amphipods. The neonate amphipods were reared under static conditions for eight days with gentle, oil-free aeration. During the holding period, dissolved oxygen ranged from 6.5 to 7.8 mg/L and temperature ranged from 23 to 24 °C. No mortality was observed in the test population 48 hours prior to exposure initiation.
- Feeding: During holding and acclimation, the freshwater amphipods were fed a combination of yeast, cereal leaves and flaked fish food suspension (YCT) on a daily basis. The day the test population was isolated, a small amount of 100 mg/mL flaked fish food suspension and Ankistrodesmus falcatus, a unicellular green algae, was added to the isolation vessels as a supplemental food source. No pesticides, PCBs and toxic metals have been detected in analysed food samples.

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

10 d

Exposure phase:

total exposure duration

Hardness:

52 - 72 mg/L as CaCO3

Test temperature:

22 - 23 °C. Continuous measurement in an auxiliary vessel maintained in the water bath used to house the test vessels established a temperature range of 22 to 24 ºC.

pH:

6.6 - 7.1

Dissolved oxygen:

4.5 - 7 .8 mg/L as oxygen

Ammonia:

- Day 0: ≤ 0.10 - 0.49 mg/L as nitrogen
- Day 10: 0.47 - 1.1 mg/L as nitrogen

Conductivity:

460 - 480 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 6.3, 13, 25, 50 and 100 mg cation/kg sediment dry weight), equivalent to 0 (control), 8.7, 18, 35, 69, and 140 mg pure substance/kg sediment dry weight.
- Mean measured concentrations sediment (t=0d): - Mean measured concentrations sediment (t=10d): Very low concentrations were detected in pore and overlying water. See 'Any other information on materials methods incl. tables' for tabulated overviews of the analytical results.

Details on test conditions:

TEST SYSTEM
- Test container: 300-mL glass beakers which were chemically cleaned prior to use and rinsed several times using tap water. Each test vessel had a hole cut on the top edge of the beaker which was covered with 40-mesh Nitex® screen for drainage.
- Sediment volume: Each vessel contained 100 mL (approximately 4-cm layer) of sediment (equivalent to 129 g wet weight per vessel or 58.9 g dry weight per vessel)
- Weight of wet sediment with and without pore water:
- Overlying water volume: 175 mL of overlying water.

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 11. Eleven replicates were maintained for each test concentration and the controls. Eight replicates (A through H) were used to evaluate the biological response (survival and growth) of the test organisms. Three replicates (I through K) were maintained for the purpose of chemical analysis.
- No. of replicates per control / vehicle control: 11
- Feeding regime: During the 10-day exposure, each replicate test vessel received 1.0 mL of YCT daily.

EXPOSURE INITIATION
At exposure initiation, amphipods (eight days old) were added impartially to an intermediate set of beakers by adding no more than two amphipods to each beaker until all beakers contained two amphipods. This process of systematically adding two additional amphipods to each beaker was repeated until each beaker contained ten amphipods. The exposure was initiated when each intermediate beaker of ten amphipods was added to each respective test vessel. Measurement of dry weight was performed on a subpopulation of twenty amphipods at exposure initiation and the dry weight was determined to be 0.020 mg dry weight per amphipod.

RENEWAL OF OVERLYING WATER
During the 10-day definitive toxicity test, the overlying water was renewed by adding two volume additions (i.e., 350 mL) per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system. The intermittent delivery system was calibrated to provide 1 L of water per cycle to the water-distribution system (Figure 1), which subsequently provided 50 mL of water per cycle to each replicate test vessel. The water delivery system cycled approximately 7 times per day (once every 3.4 hours), providing approximately 350 mL per vessel every 24 hours.
The calibration of the overlying water renewal system was checked prior to exposure initiation and confirmed at exposure termination. If there was any indication during the toxicity test that the renewal system calibration had changed (e.g., system malfunction or unexplained differences in dissolved oxygen concentration or temperature in the test vessels), the calibration of the necessary renewal system components were checked. During the definitive test, the renewal system was visually inspected at least twice daily. A complete check of intermittent delivery system functioning was made once daily.

OVERLYING WATER CHARACTERISTCS
The source of overlying water used during this study was laboratory well water. The water used during the definitive exposure was characterized as having total hardness and total alkalinity ranges as calcium carbonate (CaCO3) of 68 to 80 mg/L and 21 to 22 mg/L, respectively, a pH range of 7.0 to 7.1, and a conductivity range of 370 to 440 μS/cm. Representative samples of the overlying water source were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed in agreement with ASTM guidelines (2002). Amphipod cultures are maintained in water from the same source as the water utilized during this study and have successfully survived and reproduced over multiple generations. The acceptable performance of the amphipod cultures, in combination with the previously mentioned analyses, confirmed the acceptability of this overlying water for use during sediment toxicity testing.

NATURAL SEDIMENT
Natural, freshwater sediment was the substrate used in the exposure. The natural sediment used in the toxicity test was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to use and characterization, the sediment was wet pressed through a 2.0-mm sieve to remove large particles and indigenous organisms. The sediment used in this study was characterized as having a percent organic carbon of 4.1%, a particle size distribution of 89% sand, 10% silt and 1% clay and a pH of 5.4. A percent solids value of 45.56% and a pore water ammonia (as nitrogen) concentration of 3.1 mg/L were determined by the test facility.

ALLOCATION OF SEDIMENT TO TEST VESSELS
One day prior to exposure initiation (day -1), the treated and control sediments were allocated to each treatment or control vessel (100 mL per vessel). Overlying water was gently added to each vessel and then each vessel was placed under the renewal system. A turbulence reducer, consisting of a modified plastic disk, was used to minimize the disruption of the sediment layer during the introduction of overlying water on test day -1.

WATER QUALITY MEASUREMENTS
At exposure initiation (day 0) and termination (day 10), temperature, dissolved oxygen concentration and pH were measured in each replicate vessel of each treatment level and control used for biological monitoring (A through H) during the 10-day exposure. On test days 1 through 9, temperature and dissolved oxygen were measured daily in one alternating replicate of each treatment level and control. In addition, the temperature was continuously monitored in an auxiliary vessel in the temperature controlled water bath used to house the test vessels throughout the study using a VWR minimum/maximum thermometer. Total hardness, alkalinity, conductivity and the total ammonia of the test solutions were determined at exposure initiation and at exposure termination in a composite sample (replicates A through H).

The dissolved oxygen concentration and daily temperature were measured using a Yellow Springs Instrument (YSI) Model No. DO200A, Model No. 550A or Model No. PRO 20 dissolved oxygen meter/temperature probe. The pH was measured using a YSI Model pH100 pH meter. Total hardness and alkalinity of the test solutions at exposure initiation and termination were determined. Conductivity was monitored with a YSI Model No. 3100-115V conductivity-salinity meter. The concentration of total ammonia was determined using an Orion (Model 4-Star) meter in combination with an Orion Ammonia Electrode Model.

OTHER TEST CONDITIONS
- Light quality: The test area was illuminated with fluorescent bulbs.
- Photoperiod: 16 hours light and 8 hours darkness.
- Light intensity: 830 - 830 lux . Light intensity was measured using a VWR Traceable light meter.

EFFECT PARAMETERS MEASURED: mortality, sub-lethal effects
Following the addition of amphipods, all vessels were examined at exposure initiation (day 0) and daily thereafter, until exposure termination (day 10). Observations of mortality and abnormal behavior were made and the physical characteristics of the test solutions were recorded. At exposure termination (day 10), the remaining sediment was sieved using fine mesh fish nets (approximately 0.25 mm mesh opening) to remove all surviving amphipods and the total number and growth (expressed as dry weight) of surviving amphipods was determined for each test vessel. Growth of the surviving amphipods was determined by pooling all surviving amphipods from each replicate and drying at approximately 37 to 73 ºC (deviation, see 'Any other information on materials and methods incl. tables') for approximately 24 hours. The pooled amphipods were then weighed on a calibrated analytical balance to the nearest 0.01 mg.

Reference substance (positive control):

no

Key result

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

53.9 mg/kg sediment dw

Nominal / measured:

nominal

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:

10 d

Dose descriptor:

LC50

Effect conc.:

39 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

mortality

Remarks on result:

other: 95% C.I.: 36 - 41 mg cation/kg sediment dw

Remarks:

Original value as presented in the study report.

Details on results:

A summary of survival and growth data at termination of the sediment exposure is presented in 'Any other information on results incl. tables'.
- Controls: Following 10 days of exposure, amphipod survival in the control averaged 99%. During the same period, amphipod growth in the control averaged 0.18 mg dry weight per amphipod.
- Survival: At exposure termination (test day 10), survival observed among amphipods in the 6.3, 13, 25, 50 and 100 mg test substance cation/kg sediment dry weight treatment levels averaged 96, 100, 95, 16 and 0%, respectively.
- Growth: At exposure termination, growth observed among amphipods in the 6.3, 13, 25 and 50 mg test substance cation/kg sediment dry weight treatment levels was 0.15, 0.17, 0.16 and 0.11 mg dry weight per amphipod.

Table: Amphipod survival and growth (dry weight) in each replicate vessel at exposure termination of the 10-day static-renewal exposure of freshwater amphipods to the test substance applied to sediment

Nominal Sediment Concentration (mg test substance cation/kg sediment dry weight)	Test Day 10
	Replicate	Mean Percent Sutvival	Mean Dry Weight Per Amphipod (mg)
Control	A	100	0.16
	B	100	0.20
	C	100	0.17
	D	100	0.18
	E	100	0.17
	F	100	0.19
	G	100	0.20
	H	90	0.16
6.3	A	90	0.16
	B	90	0.16
	C	100	0.09
	D	100	0.15
	E	100	0.14
	F	100	0.17
	G	100	0.15
	H	90	0.16
13	A	100	0.17
	B	100	0.16
	C	100	0.19
	D	100	0.16
	E	100	0.11
	F	100	0.17
	G	100	0.18
	H	100	0.18
25	A	90	0.17
	B	100	0.18
	C	100	0.13
	D	100	0.16
	E	100	0.17
	F	100	0.16
	G	80	0.16
	H	90	0.15
50	A	0	NA(a)
	B	0	NA
	C	20	0.15
	D	10	0.20
	E	30	0.10
	F	40	0.08
	G	20	0.07
	H	10	0.07
100	A	0	NA
	B	0	NA
	C	0	NA
	D	0	NA
	E	0	NA
	F	0	NA
	G	0	NA
	H	0	NA

a) NA = Not Applicable.

 

Table: Mean percent survival and mean dry weight during the 10-day static-renewal exposure of amphipods to the test substance applied to sediment

Nominal Sediment Concentration (mg test substance cation/kg sediment dry weight)	Test Day 10
	Mean Percent Survival (SD)(a)	Mean Dry Weight Per Amphipod in mg (SD)
Control	99 (4)	0.18 (0.02)
6.3	96 (5)	0.15(b)(0.02)
13	100 (0)	0.17 (0.02)
25	95 (8)	0.16 (0.02)
50	16(c)(14)	0.11 (d)(0.05)
100	0(c)(0)	NA(e)

a) SD = Standard Deviation.

b) Significantly reduced compared to the control, based on Steel’s Many-One Rank Sum Test. Due to the lack of a significant effect at the next two higher treatment levels, the effect observed at this treatment level was not considered to be toxicant related.

c) Significantly reduced compared to the control, based on Steel’s Many-One Rank Sum Test.

d) Treatment level was excluded from further statistical analysis of all endpoints for the purposes of determining NOEC and LOEC values due to the effects on survival at day 10.

e) NA = Not Applicable.

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species: (100/72.4) x 39 mg test substance cation/L = 53.9 mg pure test substance/L

Validity criteria fulfilled:

yes

Conclusions:

The 10-d LC50 value was determined to be 39 mg test substance cation/kg dw. This value corresponds to a recalculated value of 53.9 mg pure substance/kg dry weight.

Executive summary:

The purpose of this study was to determine the effects of the test substance applied to sediment, on the survival and growth (final dry weight) of the sediment-dwelling freshwater amphipod, Hyalella azteca. The definitive toxicity test was performed under static-renewal conditions for a period of 10 days, at nominal concentrations of 6.3, 13, 25, 50 and 100 mg cation/kg sediment dry weight, equivalent to nominal test substance concentrations of ~ 8.7, 18, 35, 69, and 140 mg pure substance/kg sediment dry weight. Nominal sediment concentrations were achieved by spiking sediment with a combination of technical grade paraquat dichloride and radiolabeled test substance. Exposure concentrations of [14C]-labelled cation test substance (as total radioactive residue, TRR) in sediment, pore water and overlying water were analyzed on days 0 and 10 by liquid scintillation counting (LSC); sediment samples were first combusted and then measured using LSC. In addition, sediment and overlying water samples of the lowest and highest treatment levels were analyzed by high performance liquid chromatography with radiometric detection (HPLC/RAM) as a chemical-specific analysis on test days 0 and 10. Measured [14C]-labelled test substance cation concentrations and the ratio of radiolabeled to nonradiolabeled test material spiked in each treatment level were used to calculate total cation concentrations present in sediment, pore water, and overlying water at each interval. The results of this study are presented based on nominal sediment concentrations as mg test substance cation/kg sediment dry weight. Based on the results, the 10-d LC50 value was determined to be 39 mg test substance cation/kg dw. This value corresponds to a recalculated value of 53.9 mg pure substance/kg dry weight.

Reference 3

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 Nov 2014 to 17 Nov 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1740 (Whole Sediment Acute Toxicity of Invertebrates, marine)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

See 'Details on analytical methods'

Vehicle:

no

Details on sediment and application:

RADIOLABELED STOCK SOLUTION PREPARATIONS
A primary radiolabeled stock solution was prepared by quantitatively transferring the entire amount of test substance to a Nalgene(TM) container and diluting it to a final volume of 11 mL with purified reagent water. Duplicate 10.0-μL aliquots of the stock solution were then transferred to glass vials containing 10 mL of Ultima Gold cocktail and mixed well prior to being assayed by liquid scintillation counting (LSC). Based on this analysis and the Study Sponsor supplied specific activity of 202.3 μCi/mg (449,106 dpm/μg as test substance and 620,312 dpm/μg as test substance cation), the stock solution was determined to have a concentration of 0.960 mg test substance/mL.

NONRADIOLABELED STOCK SOLUTION PREPARATION
A 25 mg test substance/mL primary nonradiolabeled stock solution was prepared by placing 0.6292 g (0.6254 g as active ingredient; 99.4% a.i.) of the test substance into a volumetric flask and bringing it to a final volume of 25 mL with deionized water. The stock solution was observed to be light brown in color with no visible undissolved test substance following preparation. This primary nonradiolabeled stock solution was used to prepare the dosing solutions.

SEDIMENT DOSING STOCK SOLUTIONS
Dosing stock solutions for all test concentrations were prepared by adding appropriate volumes of both radiolabeled and nonradiolabeled primary stock solutions to a 10-mL volumetric stock. Dosing stock solutions for all test concentrations were then brought to a final volume of 10 mL with deionized water.

APPLICATION OF THE TEST SUBSTANCE TO SEDIMENT
A jar-rolling technique was used to apply the test substance to the sediment. The appropriate volume of each dosing stock solution was applied directly to 2.0 kg of wet sediment (0.7046 kg total dry weight based on a percent solid value of 35.23%) in a 4-L glass jar. The jars were sealed and positioned horizontally on the rolling mill. Each jar was then rolled for four hours at room temperature at approximately 15 rpm. Following four hours of rolling, the jars were stored upright at 2 to 8 ºC. The dosed sediments were allowed to equilibrate for a 20-day period in the refrigerator. The length of the equilibration period used was based on partitioning data previously collected for a similar compound, applied to sediment. Because of the similar chemical characteristics of these two compounds, it was determined that a similar equilibration period of spiked sediments was appropriate. Once a week during the 20-day equilibration period and prior to addition into the replicate test vessels, the jars were mixed on the rolling mill for an additional two hours at room temperature to ensure the sediment was homogeneous.
A control sample was prepared in a similar manner as the treated sediment by adding 9 mL of deionized water, containing no test substance, to 2.0 kg of wet sediment and processed in a similar manner as the treated sediments.

Test organisms (species):

Leptocheirus plumulosus

Details on test organisms:

TEST ORGANISM
- Common name: marine amphipod
- Source: The amphipods used during this study were obtained from Chesapeake Cultures, Inc., Hayes, Virginia.
- Age and size: The marine amphipods used during this study were approximately the same size and age (juvenile amphipods that pass through a 1.0-mm sieve and are retained on a 0.50-mm sieve correlating to amphipods which range from 2.0 to 4.0 mm in length)
- Feeding during test: Yes, see 'Details on test conditions'

HOLDING
- Environmental conditions: Amphipods used in the exposure were acclimated to test conditions for 48 hours prior to exposure initiation. During the acclimation period, amphipods were maintained in two 2-L glass beakers containing approximately 1.8 L of 20‰ natural, filtered seawater. The culture water was from the same source as that which was used as overlying water during the test. Measured water quality parameters determined that dissolved oxygen concentration ranged from 6.5 to 7.3 mg/L and temperature ranged from 24 to 26 ºC during the holding period.
- Feeding: During the holding period, the amphipods were fed a flaked fish food suspension, at a rate providing adequate nutrition to sustain the isolated population. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed. Based on these analyses, the food sources were considered to be of acceptable quality since analyte concentrations were below levels of concern. No mortality was observed in the test population 48 hours prior to exposure initiation.

Study type:

laboratory study

Test type:

semi-static

Water media type:

saltwater

Type of sediment:

natural sediment

Remarks:

Collected fom Sequim Bay, Sequim, Washington

Limit test:

no

Duration:

10 d

Exposure phase:

total exposure duration

Test temperature:

24 - 26 °C. Continuous measurement in an auxiliary vessel maintained in the water bath used to house the test vessels established a temperature range of 23 to 26 ºC.

pH:

7.8 - 8.6

Dissolved oxygen:

6.1 - 7.5 mg/L as oxygen

Salinity:

2.0 - 2.2%. based on daily measurements of each replicate vessel in all treatment levels and the control at exposure initiation and termination, and from one alternating replicate vessel on test day 1 through 9.

Ammonia:

- Day 0: 1.6 - 2.2 mg/L as nitrogen
- Day 10: 0.74 - 1.6 mg/L as nitrogen

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 6.3, 13, 25, 50 and 100 mg cation/kg sediment dry weight), equivalent to 0 (control), 8.7, 18, 35, 69, and 140 mg pure substance/kg sediment dry weight.
- Mean measured concentrations sediment (t=0d): - Mean measured concentrations sediment (t=10d):

Details on test conditions:

TEST SYSTEM
- Test container: The test vessels used in the static test were 1000-mL glass beakers which were chemically cleaned prior to the exposure use and rinsed several times using tap water.
- Sediment volume: Each vessel contained 175 mL (approximately 2-cm layer) of sediment (equivalent to 195 g wet weight per vessel or 68.7 g dry weight per vessel)
- Overlying water volume: 175 mL of overlying water. The total overlying water/sediment volume was maintained at approximately 900 mL.

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 9. Nine replicates were maintained for each test concentration and the controls. Five replicates (A through E) were used to evaluate the biological response of the test organisms. The remaining four replicates (F through I) were maintained for the purpose of chemical analysis and pore water quality monitoring. Twenty amphipods were added to each replicate vessel, a total of 100 amphipods per concentration or control for the replicates maintained for monitoring the biological response. The additional replicates for analytical or water quality measurements were maintained under the same conditions and contained test organisms, with the exception of replicates F and G which were sampled on day 0 prior to the addition of organisms. The additional replicates for analytical or water quality measurments were not used to evaluate the biological response of the test organisms.
- No. of replicates per control / vehicle control: 9
- Feeding regime: Test organisms were not fed during the exposure.

EXPOSURE INITIATION
Amphipods were added impartially to the replicate vessels by adding no more than five amphipods to each vessel until all replicates contained five amphipods. This procedure was repeated until each replicate contained twenty amphipods, at which point the addition of the test organisms to the vessels was completed.

OVERLYING WATER CHARACTERISTCS
The source of overlying water used during this study was natural, filtered seawater (salinity range of 30 to 31‰ and pH range of 7.8 to 8.2). Seawater was pumped from the Cape Cod Canal, Bourne, Massachusetts from about 4 meters offshore at a depth of approximately 0.5 meters. The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 3400-L fiberglass holding tank. In the laboratory, the seawater was recirculated within an epoxy-coated concrete holding reservoir prior to use. The seawater was pumped under constant pressure through PVC pipes to a laboratory holding reservoir. Prior to use in this study, the seawater was adjusted with laboratory well water resulting in a salinity ranging from 20 to 21‰ and a pH ranging from 7.7 to 8.0. Representative samples of the water source were analyzed for the presence of pesticides, PCBs and metals. None of these compounds were detected in any of the water samples analyzed in agreement with ASTM guidelines (2002). These analyses confirmed the acceptability of this overlying water for use during the conduct of the exposure.

SEDIMENT
The sediment used during this study was collected from Sequim Bay, Sequim, Washington. Prior to use and characterization, the sediment was wet pressed through a 0.25-mm sieve to remove large particles and any indigenous organisms. The sediment used in this study was characterized by Agvise Laboratories, Northwood, North Dakota, as having a percent organic carbon of 3.9%, a particle size distribution of 35% sand, 40% silt and 25% clay, a pH of 7.7, and a percent moisture at 1/3 bar (water holding capacity) of 75.6%. A percent solids of 35.23% was determined. A sample of the sediment pore water was generated from the control sediment prior to exposure initiation and was found to have a measured ammonia concentration of 15.1 mg/L as nitrogen, which is below the guideline recommended limit of 60 mg/L. A representative sample of the sediment source was analyzed for the presence of pesticides, PCBs and toxic metals. None of these compounds were found detected at concentrations that would be considered to have an adverse impact on the results of the test.

ALLOCATION OF SEDIMENT TO TEST VESSELS
One day prior to exposure initiation (day -1), the treated and control sediments (175 mL per vessel) were allocated to the nine replicate vessels per treatment or control. Overlying water was gently added to each vessel and then each vessel was placed in the water bath. A turbulence reducer, composed of a modified plastic disk, was used to minimize the disruption of the sediment layer during the introduction of overlying water.

OVERLYING WATER QUALITY MEASUREMENTS
At exposure initiation (day 0) and termination (day 10), temperature, pH, dissolved oxygen concentration and salinity were measured in the overlying water of each replicate vessel of each treatment level and control used for biological monitoring (A through E) during the 10-day exposure. On test days 1 through 9, temperature, dissolved oxygen, pH and salinity were measured in one alternating replicate from each treatment level and control each day. In addition, the temperature was continuously monitored in an auxiliary vessel in the temperature controlled water bath used to house the test vessels throughout the study using a VWR minimum/maximum thermometer. Total ammonia concentration of the overlying water was determined at exposure initiation and at exposure termination in a composite sample of overlying water from each treatment and control group.
The dissolved oxygen concentration and daily temperature were measured using a Yellow Springs Instrument (YSI) Model 550A or Model Pro20 dissolved oxygen meter/temperature probe. The pH was measured using a YSI Model pH100 pH meter. The concentration of total ammonia was determined using an Orion 4-Star meter in combination with an Orion Ammonia Electrode Model 9512HPBNWP. Salinity was measured using a VitalSine Model SR-6 refractometer.

PORE WATER QUALITY MEASUREMENTS
At exposure initiation and again at termination, salinity, temperature, pH and total ammonia (as nitrogen) concentration was measured in a pore water sample of each treatment level and control. The pH was measured using a YSI Model pH100 pH meter. The concentration of total ammonia was determined using an Orion 4-Star meter in combination with an Orion Ammonia Electrode Model 9512HPBNWP. Salinity was measured using a VitalSine Model SR-6 refractometer.

OTHER TEST CONDITIONS
- Light quality: The test area was illuminated with fluorescent bulbs.
- Photoperiod: 16 hours light and 8 hours darkness.
- Light intensity: 540 - 860 lux . Light intensity was measured using a VWR Traceable light meter.

EFFECT PARAMETERS MEASURED: mortality, sub-lethal effects
All vessels were examined at exposure initiation and daily thereafter, until exposure termination (day 10). Observations of mortality and abnormal behavior were made and the physical characteristics of the test solutions were recorded. At exposure termination (day 10), the total number of surviving amphipods was determined in each test vessel by sieving the entire volume of sediment through a fine mesh net (≤ 0.50 mm mesh opening) to remove all surviving amphipods. Missing organisms or those failing to respond to gentle prodding (i.e., neuromuscular twitch of pleopods or antennae) were recorded as dead.

Reference substance (positive control):

no

Key result

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

> 138 mg/kg sediment dw

Nominal / measured:

nominal

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:

10 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

mortality

Remarks on result:

other: Original value as presented in the study report.

Details on results:

A summary of survival and growth data at termination of the sediment exposure is presented in 'Any other information on results incl. tables'.
- Controls: Following 10 days of exposure, amphipod survival in the control averaged 99%. During the same period, amphipod growth in the control averaged 0.18 mg dry weight per amphipod.
- Survival: At exposure termination (test day 10), survival observed among amphipods in the 6.3, 13, 25, 50 and 100 mg test substance cation/kg sediment dry weight treatment levels averaged 96, 100, 95, 16 and 0%, respectively.
- Growth: At exposure termination, growth observed among amphipods in the 6.3, 13, 25 and 50 mg test substance cation/kg sediment dry weight treatment levels was 0.15, 0.17, 0.16 and 0.11 mg dry weight per amphipod.


Following 10 days of exposure, amphipod survival in the control averaged 98%. Amphipod survival met the minimum standard criteria established by the OCSPP Guideline (i.e., ≥ 90% survival). As demonstrated by the control organism performance, the exposure system provided test conditions that were appropriate for promoting acceptable survival of Leptocheirus plumulosus during the exposure duration.

Table: Amphipod survival and growth (dry weight) in each replicate vessel at exposure termination of the 10-day static-renewal exposure of marine amphipods to the test substance applied to sediment

Nominal concentration (mg test substance cation/kg sediment dry weight)	Replicate	# Surviving Amphipods	Percent Survival	Mean Percent Survival (SD(a))
Control	A	20	100	98 (3)
	B	20	100
	C	19	95
	D	19	95
	E	20	100
6.3	A	18	90	98 (4)
	B	20	100
	C	20	100
	D	20	100
	E	20	95
13	A	19	95	98 (3)
	B	20	100
	C	20	100
	D	20	100
	E	19	95
25	A	20	100	88 (27)
	B	20	100
	C	8	40
	D	20	100
	E	20	100
50	A	20	100	98 (3)
	B	19	95
	C	20	100
	D	19	95
	E	20	100
100	A	19	95	99 (2)
	B	20	100
	C	20	100
	D	20	100
	E	20	100

a) NA = Not Applicable.

 

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species: (100/72.4) x 100 mg test substance cation/L = 138 mg pure test substance/L

Validity criteria fulfilled:

yes

Conclusions:

The 10-d LC50 value was determined to be >100 mg/kg test substance cation/kg sediment dw. This value corresponds to a recalculated value of >138 mg pure test substance/kg sediment dw.

Executive summary:

The purpose of this study was to determine the effects of the test substance applied to sediment, on the survival of the sediment-dwelling marine amphipod, Leptocheirus plumulosus. The definitive toxicity test was performed under static conditions for a period of 10 days, at nominal test substance cation concentrations of 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight, equivalent to nominal pure test substance concentrations of 8.7, 18, 35, 69, and 140 mg/kg sediment dry weight. Exposure concentrations of radiolabelled test substance cation (as total radioactive residue, TRR) in sediment, pore water and overlying water were analyzed on days 0 and 10 by liquid scintillation counting (LSC); sediment samples were first combusted and then measured using LSC. In addition, sediment and overlying water samples of the lowest and highest treatment levels were analyzed by high performance liquid chromatography with radiometric detection (HPLC/RAM) as a chemical-specific analysis on test days 0 and 10. Measured radiolabelled test substance cation concentrations and the ratio of radiolabeled to nonradiolabeled test material spiked in each treatment level were used to calculate total test substance cation concentrations present in sediment, pore water and overlying water at each interval. No significant effects were observed up to the highest test concentration. Therefore, the 10-d LC50 value was determined to be >100 mg/kg test substance cation/kg sediment dw. This value corresponds to a recalculated value of >138 mg pure test substance/kg sediment dw.

Description of key information

Freshwater: The 10-d LC50 value is 53.9 mg pure substance/kg sediment, determined in a H. azteca short-term toxicity test, OPPTS 850.1735, Bradley 2015

Freshwater: The 21-d NOEC value is 138.1 mg pure substance/kg sediment, determined in a C. riparius emergence test, ASTM E1381, Hamer 1998

 

Saltwater: The 10-d LC50 value is >138.1 mg pure substance/kg sediment, determined in Leptocheirus plumulosus short-term toxicity test, OPPTS 850.1740, Bradley 2015b

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:

53.9 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

138.1 mg/kg sediment dw

Additional information

Table: Overview of the available toxicity data on sediment-dwelling organisms

Species	Guideline/ GLP	Endpoint	Effect value	Comment	Reference
Chironomus riparius	ASTM E1383 and SETAC Recommendations on sediment testing/ GLP	21-d NOEC	138.1 mg pure substance/kg sediment dw (recalculated)	No effects observed at the limit concentration. Partition of the test substance to the overlying water compartment was negligible.	Hamer 1998
Hyalella Azteca	OPPTS 850.1735/ GLP	10-d LC50	53.9 mg pure substance/kg sediment dw (recalculated)		Bradley 2015
Leptocheirus plumulosus (marine amphipod)	OPPTS 850.1740/ GLP	10-d LC50	>138 mg pure substance/kg sediment dw (recalculated)	No significant effects observed up to the highest test concentration.	Bradley 2015b