1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Nov 2014 - 29 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 235 (Chironomus sp., Acute Immobilisation Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Details on sampling:

WATER
- Concentrations: control, solvent control, 8.00, 16.0, 32.0, 64.0, 124 and 256 µg a.s./L
- Sampling interval: test start (0 h) and test end (48 h); Samples (10 mL) from test start were sampled from the prepared volume of each test treatment level. For sampling of aged test media, the contents of all six replicate vessels of day 2 were combined and were then submitted for analyses.
- Sample storage before analysis: The samples were stored in a freezer until they were analysed.

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on sediment and application:

PREPARATION OF SPIKED WATER
- Details of spiking: Stock solution: 142.9 mg test item added to 50 mL Dimethylformamide (DMF); well agitated on a magnetic stirrer for 5 minutes; used for following dilution series with Dimethylformamide (S2 – S6) were prepared and by adding appropriate aliquots of these stock solutions to the test medium
- Controls: yes, control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: non-biting midge
- Source: University of Frankfurt am Main (Germany), received: transferred in July 2006 to the lab and kept since then
- Details on collection: 2 - 4 egg masses are placed into the prepared basin. After 2 to 3 weeks the adults emerge. After mating, female adults will lay egg masses on the water surface where these can be taken to start a new culture or to perform toxicity tests.
- Age of parental stock (mean and range, SD): 2 to 3 weeks
- Breeding conditions:
For breeding the midges are kept in cages (40 x 40 x 40 cm), with a gauze on each side of the cage. A basin is set on the bottom of each cage.The bottom of the basins are covered with a thin layer of "Kieselgur" (silica) and a 4 - 5 cm high layer of reconstituted water according to Elendt medium (M7; prepared with deionised water; iImmediately before use in this study, the vitamin components were added by a separately deep frozen stored stock solution) and aerated gently. The culture conditions are 20 ± 2°C and 16 to 8 hours light-darkcycle (light intensity ca. 500 - 1000 lux).
- Handling of egg masses and larvae: Larvae were obtained by introducing some fresh egg masses in small dishes with test medium. On study day 0 the larvae were transferred in a randomised procedure into the test vessels (collectives of five larvae each).
- Age of animals at beginning of exposure: Two to three days after hatching the L1-larvae were transferred carefully with a blunt pipette to the test vessels.
- Feeding during test: prior to insertion of the larvae into the test vessels, a few drops of an aqueous fish food suspension (50 g Tetra Phyll®/L deion. water, refrained from the settled fraction) were applied to the culture dishes.
- Frequncy: Once
- Food type: The hatched larvae are fed with green algae and an aqueous suspension of a plant material based fish food (Tetra Phyll®).

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

48 h

Exposure phase:

total exposure duration

Test temperature:

20.3 - 20.5 °C (test start)
20.4 - 20.6 °C (test end)

pH:

7.8

Dissolved oxygen:

8.4 mg/L (test start)
8.3 to 8.4 mg/L (test end)

Ammonia:

NH4+: <0.03 mg/L (water)

Conductivity:

< 10 μS/cm

Nominal and measured concentrations:

0 (control), solvent control, 8.00, 16.0, 32.0, 64.0, 128 and 256 μg test item/L test medium (nominal)
< 0.625, < 0.625, 8.67, 17.5, 34.2, 66.3, 158 and 306 µg test item/L (based on initial measured concentration)
< 0.625, < 0.625,7.57, 16.0, 30.9, 60.9, 147 and 282 µg test item/L (based on measured concentration after Day 2)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 20 mL glass beakers
- Overlying water volume: 10 mL
- Aeration: no

EXPOSURE REGIME
- No. of organisms per container (treatment): 5
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 6
- Feeding regime: Once, prior to insertion of the larvae into the test vessels
- Type and preparation of food:queous fish food suspension (50 g Tetra Phyll®/ L deion. water, refrained from the settled fraction)
- Amount of food: a few drops

WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Elendt medium (M7), prepared with deionised water.
- Source of water (if non-standard medium): deionised water, supplied by Currenta GmbH & Co. OHG, Energie – Wasserversorgung; quality is checked at least twice a year (non-GLP data) for residues and contaminants (e.g. pesticides and heavy metals)
- Location: Leverkusen
- Alkalinity: 53.4 mg/L CaCO3
- Conductivity: below 10 μS/cm
- Particulate matter: < 5 mg/L (total solids)
- Residual chlorine: < 0.01 mg/L
- Total organic carbon: < 2 mg/L
- Chemical oxygen demand:< 5 mg/L
- Inorganic contaminants: Bor, F-: < 100 µg/L; NH4+: <0.03 mg/L; Zn,Al: 1 µg/L; As, Cr, Cu, Co, Fe, Pb, Ni: < 1 µg/L; Cd, Hg, Ag: <0.1 µg/L
- Total organic chlorine compounds: < 0.1 µg/L
- Pesticides Residue contaminants: < 0.05 µg/L

OTHER TEST CONDITIONS
- Photoperiod:16 to 8 h (light to dark)
- Light intensity: 500-1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): number of immobilised larvae; sub-lethal effects were recorded

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
Range finding study
- Test concentrations: control, solvent control, 0.01; 0.10; 1.0 and 10.0 μg a.s./L
- Results used to determine the conditions for the definitive study: Concentration range of the definitive test was based on the results of the non-GLP range-finding test

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

48.8 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mobility

Remarks on result:

other: 95 % confidence limits

Remarks:

27.8 - 68.7 µg/L

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

83.1 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mobility

Remarks on result:

other: 95 % confidence limits

Remarks:

57.2 - 113.3 µg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

230 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mobility

Remarks on result:

other: 95 % confidence limits

Remarks:

162.4 - 413.5 µg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mobility

Details on results:

- Immobile test animals at end of exposure period: 32.0 µg a.s./L=1 (of 30); 64.0 µg a.s./L=7 (of 30); 128 µg a.s./L=9 (of 30); 256 µg a.s./L=15 (of 30)
- Total mass of test animals at beginning of test: 30
- Other biological observations: immobile animal lies on the bottom of the test vessel showing reduced mobility
- Other: biological and analytical results are sumarized within the tables 1 and 2 in section "Any other information on results incl. tables".

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Dose-response test: yes; test concentrations: 0.43, 0.94, 2.07, 4.55 and 10.0 mg/L.
- ECx: The current 48 h EC50 of 2.06 mg/L, as determined in this test, meets the range defined by OECD 235 (0.50 mg/L - 3.6 mg/L)

Reported statistics and error estimates:

EC50 values and confidence intervals were calculated for the stated exposure period, using a commercial program “ToxRat Professional” using probit analysis. NOEC and LOEC were calculating follwoing Williams Multiple Sequential t-test Procedure (Alpha = 0.05, one-sided smaller)

Table 1: Acute toxicity of test item to first instar-larvae of Chironomus riparius after 48 hours (based on nominal concentrations):

Test concentration [μg a.s./L]	Exposed chironomids (=100%)	Immobility
		24 h		48 h
		n	%	n	%
Control	30	0	0	0	0
Solvent control	30	0	0	0	0
8.00	30	0	0	0	0
16.0	30	0	0	0	0
32.0	30	0	0	1	3.3
64.0	30	2	6.7	7	23.3*
128	30	1	3.3	9	30.0*
256	30	3	10.0*	15	50.0*

* statistically significant

Analytical results:

The analytical findings of analysed a.s. found in all freshly prepared test levels on day 0 in reference to nominal concentrations ranged between 104 and 123% (average 112%). In aged test levels on day 2 there were analytical findings between 95 and 115% (average 102%) of nominal. Due to the high recoveries at the beginning of the exposure and the analytical findings after 2 days, all results are based on nominal concentrations.

Table 2: Nominal and measured concentrations of active substance [μg a.s./L]

Nominal concentrations [μg a.s./L]	Day 0		Day 2
	Analysed conc. mean of two analyses each [μg a.s./L]	Percent of nominal	Analysed conc. mean of two analyses each [μg a.s./L]	Percent of nominal
Control	< 0.625	-	< 0.625	-
solvent-control	< 0.625	-	< 0.625	-
8.00	8.67	108	7.57	95
16.0	17.5	109	16.0	100
32.0	34.2	107	30.9	97
64.0	66.3	104	60.9	95
128	158*	123	147	115
256	306	120	282	120
Average		112		102

* mean of 4 analyses

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul 2015 - 24 Aug 2015

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 (draft)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: Control, Solvent Control, 100, 200, 400, 800 and 1600 µg a.i./kg (nominal)
- Sampling interval: test start (day 0) and test end (day 10)
- Sample storage before analysis: no storage

PORE WATER
- Concentrations: Control, Solvent Control, 100, 200, 400, 800 and 1600 µg a.i./kg (nominal)
- Sampling interval: test start (day 0) and test end (day 10)
- Sample storage before analysis: no storage

OVERLYING WATER
- Concentrations: Control, Solvent Control, 100, 200, 400, 800 and 1600 µg a.i./kg (nominal)
- Sampling interval: test start (day 0) and test end (day 10)
- Sample storage before analysis: no storage

Vehicle:

yes

Remarks:

Acetone

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: The dosed sand was placed in a fume hood and the acetone was allowed to evaporate for approximately one hour. The 50 g sand premixes were added to 450 g of natural sediment in 2000 mL plastic Nalgene® bottles and mixed on a rotary mixer for approximately one hour. Next, 500 g of natural sediment were added to the premixes to achieve a final weight of 1000 g.
- Method of mixing: mixed on a rotary mixer for approximately 16 h.
- Details of spiking: A volume of 10 mL of the appropriate stock solution was added to 50.0 g of sand in a labeled glass beaker and stirred with a glass stir rod until homogenous, for each concentration.
- Controls:yes, control and solvent control
- Equilibration time: 2 d prior to the introduction of the test organisms
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 10 mL of acetone to 50.0 g of sand
- Evaporation of vehicle before use: yes, for one hour

Test organisms (species):

Leptocheirus plumulosus

Details on test organisms:

TEST ORGANISM
- Common name: amphipod
- Source: Chesapeake Cultures, Inc., Hayes, Virginia, USA
- Details on collection: identity and size (2-4 mm) of the species; organisms were held for two days at the same temperature used in the test and in water from the same source as the water used during the test.
- Handling: 20 amphipods were transfer into test chambers; transfers were made below the water surface using wide-bore pipettes.
- Feeding during test: no feeding
- Food type: Gorp (mixture of TetraMin® flake food, wheat grass, rabbit food and Neonovum®)

Study type:

laboratory study

Test type:

static

Water media type:

saltwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

10 d

Exposure phase:

total exposure duration

Test temperature:

24.1 to 25.0 °C (control)
24.4 to 25.0 °C (solvent control)
24.3 to 25.0 °C (test concentrations)

pH:

8.4 to 8.7 (control)
8.3 to 8.6 (solvent control)
8.0 to 8.6 (test concentrations)

Dissolved oxygen:

7.3 to 7.4 mg/L (control)
7.1 to 7.4 mg/L (solvent control)
6.9 to 7.4 mg/L (test concentrations)

Salinity:

20 ‰ (control)
20 ‰ (solvent control)
20 to 22 ‰ (test concentrations)

Ammonia:

3.73 to 4.79 mg/L as NH3 (control)
4.80 to 5.68 mg/L as NH3 (solvent control)
1.68 to 5.17 mg/L as NH3 (test concentrations)

Nominal and measured concentrations:

control, solvent control; 100; 200; 400; 800; 1600 µg a.i./kg (nominal)
n.d., n.d., 45; 103; 199; 356 and 728 µg a.i./kg dry sediment (mean measured concentration)
< LOQ, < LOQ, 1.69, 4.08, 14.5, 25.6 and 51.0 µg a.i./L overlaying water (initial measured concentrations)
< LOQ, < LOQ, 54.3, 97.7, 211, 341and 898 µg a.i./L pore water (initial measured concentrations)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 L glass beakers
- Sediment volume: 150 mL of sediment (1.5 cm in Negative Control replicate A)
- Sediment depth: achieving a depth of approximately 1.5 cm
- Overlying water volume: 775 mL of overlying water (9.3 cm in Negative Control replicate A)
- Water depth: 9.3 cm
- Aeration: yes
- Aeration frequency and intensity: in each test chamber through a glass pipette placed 2 cm from the surface of the sediment; intensity: greater than 1 bubble/second but not so great as to disturb the sediment

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 8
- No. of replicates per control / vehicle control: 8

OVERLYING WATER CHARACTERISTCS
- Type of water: sea water
- Source of water (if non-standard medium): natural seawater
- Location: Indian River Inlet, Delaware, USA
- Filtration: a sand filter to remove particles ≥ 25 μm and filtered through 0.45 μm to remove microorganisms and fine particles and was UV sterilized.
- Salinity: approximately 20‰
- Total organic carbon: < 2 mg/L
- Pesticides, Organics: Bolstar,Guthion(Azinphos-methyl),Coumaphos,Malathio,Demeton-O,Merphos,Demeton-S,Diazinon,Methyl Parathion,Dichlorvos,Mevinphos,Disulfoton, Dursban,(Chlorpyrifos), Naled: < 4.9 µg/L;
o,p-DDT, Endosulfan Sulfate , p,p-DDD ,Endrin , p,p-DDE ,p,p-DDT ,Endrin Ketone ,o,p-DDD ,o,p-DDE ,Dieldrin , Gamma Chlordane: < 0.016 µg/L; Chlordane < 0.41µg/L; Kepone < 0.16 µg/L; Mirex < 0.041 µg/L; Endosulfan II < 0.025 µg/L; Toxaphene 0.82 µg/L
- Metals: Aluminum < 0.200 mg/L, Magnesium 682 mg/L, Antimony < 0.0200 mg/L, Manganese < 0.0050 mg/L, Arsenic < 0.0200 mg/L, Mercury < 0.00020 mg/L,, Barium 0.0062 mg/L, Nickel < 0.0100 mg/L, Beryllium < 0.0050 mg/L, Nitrate Nitrogen < 0.50 mg/L, Bromide 25.0 mg/L, Nitrite Nitrogen < 20.0 mg/L, Cadmium < 0.0050 mg/L, Potassium 231 mg/L, Calcium 242 mg/L, Selenium < 0.0200 mg/L, Chloride 12,100 mg/L, Silver < 0.0050 mg/L, Sodium 5,670 mg/L, Cobalt < 0.0050 mg/L, Sulfate 1,440 mg/L, Copper < 0.0100 mg/L, Thallium < 0.0300 mg/L, Fluoride < 0.50 mg/L, Vanadium < 0.0050 mg/L, Iron < 0.200 mg/L, Zinc < 0.0200 mg/L, Chromium < 0.0150 mg/L, Lead < 0.0150 mg/L

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Wye River, Maryland, USA

HANDLING OF NATURAL SEDIMENT
- Water depth: Mid-depth
- Storage conditions: stored refrigerated
- % sand: 95
- % silt: 3
- % clay: 2
- Colour/texture: Sand
- pH pore water: 7.8
- pH dry matter and/or whole sediment: 7.8
- Total organic carbon (%): 0.31%
- Pesticides and Organics [µg/kg]: <5.6: Aldrin; Gamma BHC – Lindane; Alpha BHC; Gamma Chlordane; Alpha Chlordane; HCB; Heptachlor Epoxide; Heptachlor; Endosulfan I; < 11: Mirex; o,p-DDD; o,p-DDE; Endosulfan II; o,p-DDT; Endosulfan Sulfate; p,p-DDD; Endrin; p,p-DDE; Endrin Aldehyde; p,p-DDT; Dieldrin; < 89: Phorate; EPN; Ronnel; Ethion; Stirophos; Ethoprop; Ethyl Parathion; Tokuthion; Famphur; Trichloronate; Fenthion; Trithion; Dursban (Chlorpyrifos); Bolstar; Coumaphos; Malathion; Demeton-O; Merphos; Demeton-S; Diazinon; Methyl Parathion; Dichlorvos; Mevinphos; Disulfoton; Naled; Guthion Azinphos-methyl); Toxaphene < 220; Fensulfothion < 270; Telodrin < 8.0; Endrin Ketone < 12; Beta BHC < 6.7; Chlordane < 110; Kepone < 47; Delta BHC < 6.0; Methoxychlor < 45
_Metals [mg/kg]: Aluminum 999; Magnesium 426; Antimony < 2.64; Manganese 106; Arsenic < 2.64; Mercury < 25.9; Barium 3.81; Nickel < 1.32; Beryllium < 0.661; Nitrate by IC (solid) < 2.0; Bromide by IC (solid) 8.4; Nitrite by IC (solid) < 26.6; Cadmium < 0.661; Potassium 255; Calcium 172 Selenium < 2.64; Chloride by IC (solid) 2,690; Silver < 0.661; Chromium 4.33; Sodium 1,530; Cobalt < 0.661; Sulfate by IC (solid) 422; Copper < 1.32; Thallium < 3.96; Fluoride by IC (solid) < 1.3; Vanadium 3.01; Iron 1,210; Zinc 4.40; Lead 1.98
- CEC: 10.6 meq / 100 g
- Base Saturation Data: Cations: Calcium 9.8% 207 ppm; Magnesium 22.1% 280 ppm; Sodium 61.7% 1500 ppm; Potassium 2.6% 107 ppm; Hydrogen 3.8% 4 ppm

OTHER TEST CONDITIONS
- Light quality: fluorescent tubes that emitted wavelengths similar to natural sunlight
- Photoperiod: 24 h of continuous light
- Light intensity: 552 lux

EFFECT PARAMETERS MEASURED
Observations of mortality and abnormal behavior were made daily during the test. Survival was determined at the end of the 10-day test period.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
Range finding study: yes
- Results used to determine the conditions for the definitive study: Test concentrations of the definitive were based on the results of the range-finding test.

Reference substance (positive control):

no

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

> 728 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: based on sediment concentration

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

>= 728 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: based on sediment concentration

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

> 728 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: based on sediment concentration

Details on results:

- Biological and Analytical results are summarized with the tables 1 and in section" Any other information on results incl tables"
- Percent survival per control and test concentrations (mean) at the end of exposure period: negative control, solvent control, 45, 103, 199, 356 and 728 μg a.i./kg treatment groups were 100, 100, 100, 100, 100, 99 and 99%, respectively
- Observation during the exposure period: Day 4: Control: one out of 20 organism in the control observed leaving the sediment or on the surface of the sediment, Day 7: one out of 20 organism at 45 µg a.i./kg climbed the wall of the test chamber, Day 3: one out of 20 organism at 199 µg a.i./kg observed leaving the sediment or on the surface of the sediment
- Observations of mortality and effects at the end of the exposure period: 199 µg a.i./kg: one out of 20 organism appeared small in size when compared to the control organisms; 356 and 728 µg a.i./kg: one out of 20 organism Missing and assumed dead

Reported statistics and error estimates:

The survival data precluded the calculation of an LC50 value. The no-observed-effect-concentration (NOEC) and the lowest-observed-effect-concentration (LOEC) were determined by visual interpretation of the dose-response pattern and statistical analyses of the survival data. The control and solvent control had 100% survival.

Table 1: Percent Survival by Replicate in a 10-Day Sediment Toxicity Test with Leptocheirus plumulosus 

Mean Measured Concentration in Sediment (μg a.i./kg)	Replicate	Percent Survival	Mean Percent Survival
Negative Control	A	100	100
	B	100
	C	100
	D	100
	E	100
Solvent Control	A	100	100
	B	100
	C	100
	D	100
	E	100
45	A	100	100
	B	100
	C	100
	D	100
	E	100
103	A	100	100
	B	100
	C	100
	D	100
	E	100
199	A	100	100
	B	100
	C	100
	D	100
	E	100
356	A	100	99
	B	95
	C	100
	D	100
	E	100
728	A	100	99
	B	100
	C	100
	D	100
	E	95

Table 2: Measured concentration of stock solution

Nominal Test Concentration (μg a.i./mL)	Sample Number (149A-252A-)	Measured Concentration1 (μg a.i./mL)	Percent of Nominal1 (%)
10	St-1	8.61	86.1
20	St-2	17.2	86.2
40	St-3	36.6	91.6
80	St-4	74.5	93.1
160	St-5	139	86.7

1 Results were generated using OpenLab version A.01.05. Manual calculations may differ slightly

Table 3: Measured Concentrations of the test item in Sediment Samples from a Sediment Toxicity Test with Leptocheirus plumulosus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-252A-)	Sampling Interval (Day)	Measured Concentration (μg a.i./kg)1,3	Percent of Nominal2,3	Mean Measured Concentration (μg a.i./kg)	Mean Percent of Nominal
0.0	S-1	0	<LOQ	--	--	--
(Negative Control)	S-9	10	<LOQ	--
0.0	S-2	0	<LOQ	--	--	--
(Solvent	Control)	S-10	10	<LOQ	--
100	S-3	0	59.3	59.3	45	45.0
	S-11	10	30.0	30.0
200	S-4	0	144	71.9	103	51.5
	S-12	10	61.2	30.6
400	S-5	0	292	73.1	199	49.8
	S-13	10	106	26.4
800	S-6	0	494	61.8	356	44.5
	S-14	10	217	27.1
1600	S-7	0	990	61.9	728	45.5
	S-15	10	466	29.1

1 The limit of quantitation (LOQ) was 0.510 μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.0200 μg a.i./L) and the dilution factor of the matrix blank samples (20.0), divided by the soil content (0.785) of the negative control from day 0.

2 Results were generated using Excel 2010 in full precision mode and Analyst 1.6. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

Table 4: Measured Concentrations of the test item in Overlying Water Samples from a Sediment Toxicity Test with Leptocheirus plumulosus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-252A-)	Sampling Interval (Day)	Measured test item Concentration (μg a.i./L)1,2
0.0	OW-1	0	<LOQ
(Negative Control)	OW-8	10	<LOQ
0.0	OW-2	0	<LOQ
(Solvent Control)	OW-9	10	<LOQ
100	OW-3	0	1.69
	OW-10	10	0.286
200	OW-4	0	4.08
	OW-11	10	1.52
400	OW-5	0	14.5*
	OW-12	10	2.06
800	OW-6	0	25.6*
	OW-13	10	7.11
1600	OW-7	0	51.0*
	OW-14	10	19.4

1 The limit of quantitation (LOQ) was 0.200 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0200 μg a.i./L) and the dilution factor of the matrix blank samples (10.0).

2 Results were generated using Analyst version 1.6. Manual calculations may differ slightly.

* Extrapolated value.

Table 5: Measured Concentrations of test item in Pore Water Samples from a Sediment Toxicity Test with Leptocheirus plumulosus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-252A-)	Sampling Interval (Day)	Measured test item Concentration (μg a.i./L)1,2
0.0 (Negative Control)	PW-1	0	< LOQ
0.0 (Solvent Control)	PW-2	0	< LOQ
	PW-9	10	< LOQ
100	PW-3	0	54.3
	PW-10	10	13.7
200	PW-4	0	97.7
	PW-11	10	48.9
400	PW-5	0	211
	PW-12	10	76.8
800	PW-6	0	341
	PW-13	10	134
1600	PW-7	0	898
	PW-14	10	207

1 The limit of quantitation (LOQ) was 0.200 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0200 μg a.i./L) and the dilution factor of the matrix blank samples (10.0).

2 Results were generated using Analyst version 1.6. Manual calculations may differ slightly.

Note: For the negative control sample on Day 10, there was an insufficient volume of pore water.

Table 6: Measured Concentrations of metabolite in Sediment Samples from a Sediment Toxicity Test with Leptocheirus plumulosus

Nominal test item Concentration (μg a.i./kg)	Sample Number (149A-252A-)	Sampling Interval (Day)	Measured metabolite Concentration (μg a.i./kg)1,3
0.0	S-1M	0	< LOQ
(Negative Control)	8M-SED	10	< LOQ
0.0	S-2M	0	< LOQ
(Solvent Control)	9M-SED	10	< LOQ
100	S-3M	0	3.95
	10M-SED	10	< LOQ
200	S-4M	0	7.93
	11M-SED	10	< LOQ
400	S-5M	0	16.1
	12M-SED	10	69.3
800	S-6M	0	27.9*
	13M-SED	10	189
1600	S-7M	0	65.4
	14M-SED	10	549

1 The limit of quantitation (LOQ) was 1.27 μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.0500 μg a.i./L) and the dilution factor of the negative control samples (20.0), divided by the soil content (0.785) of the negative control sediment sample on Day 0.

2 Results were generated using Excel 2010 in full precision mode and Analyst 1.4.1. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

* Extrapolated value.

Table 7: Measured Concentrations of the metabolite in Overlying Water Samples from a Sediment Toxicity Test with Leptocheirus plumulosus

Nominal Test Concentration      (μg a.i./kg)	Sample Number (149A-252A-)	Sampling Interval (Day)	Measured metabolite Concentration (μg a.i./L)1,2
0.0	OW-1M	0	<LOQ
(Negative Control)	8M-OW	10	<LOQ
0.0	OW-2M	0	<LOQ
(Solvent Control)	9M-OW	10	<LOQ
100	OW-3M	0	1.09
	10M-OW	10	1.21
200	OW-4M	0	1.77
	11M-OW	10	3.22
400	OW-5M	0	<LOQ
	12M-OW	10	5.32
800	OW-6M	0	5.95
	13M-OW	10	9.52
1600	OW-7M	0	10.6*
	14M-OW	10	14.8*

1 The limit of quantitation (LOQ) was 0.500 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0500 μg a.i./L) and the dilution factor of the negative control samples (10.0).

2 Results were generated using Analyst version 1.4.1. Manual calculations may differ slightly.

* Extrapolated value.

Table 8: Measured Concentrations of metabolite in Pore Water Samples from a Sediment Toxicity Test with Leptocheirus plumulosus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-252A-)	Sampling Interval (Day)	Measured metabolite Concentration (μg a.i./L)1,2
0.0	PW-1M	0	< LOQ
(Negative Control)
0.0	PW-2M	0	< LOQ
(Solvent Control)	9M-PW	10	< LOQ
100	PW-3M	0	< LOQ
	10M-PW	10	< LOQ
200	PW-4M	0	0.491
	11M-PW	10	0.628
400	PW-5M	0	0.988
	12M-PW	10	1.20
800	PW-6M	0	2.24
	13M-PW	10	2.10
1600	PW-7M	0	3.81
	14M-PW	10	3.33

1 The limit of quantitation (LOQ) was 0.500 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0500 μg a.i./L) and the dilution factor of the negative control sample (10.0).

2 Results were generated using Analyst version 1.6. Manual calculations may differ slightly.

Note: For the negative control sample on Day 10, there was an insufficient volume of pore water.

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Nov 2013 - 17 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Version / remarks:

adopted 13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen

Analytical monitoring:

yes

Details on sampling:

Samples were taken from sediment, overlying water and pore water.
The concentration of the test item and its metabolite were analysed all compartiments sediment, overlying water and pore water.

SEDIMENT
- Concentrations: Control, 2.50; 5.0; 10.0; 40.0 and 80.0 µg a.s./kg dw sed
- Sampling interval: day -2 and day 0 (directly before inserting the larvae), day 4, day 8 and day 28 after insertion of the larvae
- Sample storage before analysis: deep-frozen at ≤ -18 °C

PORE WATER
- Concentrations: Control, 2.50; 5.0; 10.0; 40.0 and 80.0 µg a.s./kg dw sed
- Sampling interval: day 0 (directly before inserting the larvae), day 4, day 8 and day 28 after insertion of the larvae
- Sample storage before analysis: deep-frozen at ≤ -18 °C

OVERLYING WATER
- Concentrations: Control, 2.50; 5.0; 10.0; 40.0 and 80.0 µg a.s./kg dw sed
- Sampling interval:day 0 (directly before inserting the larvae), day 4, day 8 and day 28 after insertion of the larvae
- Sample storage before analysis:deep-frozen at ≤ -18 °C

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: 75.0% quartz sand (grain size: 91.4% of the particles were in the range of 50-200 μm, originated form Quarzwerke GmbH D-50226 Frechen, Germany), 4% sphagnum moss peat (pH 3.5, particle size of ≤ 1 mm, air dried, originated from Erdenwerk Gregor Ziegler GmbH D-95703 Plößberg, Germany), 20% kaolinite (kaolinite content of 30.2%, pH value 5.5 – 7.5, "Kaolin W", from Erbslöh / Geisenheim, Germany), 1% calcium carbonate (chemical pure, Merck) to adjust the pH value to 7 ± 0.5 (figures refer to dry weight) and 48% deionised water.
- Method of mixing: The formulated sand was then mixed with the suitable amounts of sediment per test concentration by feed mixer. The bottom of the test vessels were covered with a 1.5 cm layer of wet sediment (corresponding to about 140 g). M7 medium was added as test water to the sediment layer. To avoid a separation of the sediment ingredients during this procedure, the sediment was covered by a sheet, and the test water poured slowly into the vessels (final test water volume: 0.38 L). The sheet was removed carefully thereafter.
- Details of spiking: Two days before the start of the study (insertion of the larvae) spiked sediments of the test concentrations were prepared by addition of solutions of the test substance directly into the sediment. Stock solutions of the test substance dissolved in acetone were used. Suitable amounts of these stock solutions were mixed with suitable amounts of fine quartz sand for each test concentration (10 g / test vessel).
- Equilibration time: For equilibration the sediment had been prepared 7 days before spiking.
- Equilibration conditions: In case of a loss of water by evaporation, the test vessels were refilled with deionised water up to the mark, which indicates a volume of 0.38 L overlying water (typically once a week).
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: The solvent was allowed to evaporate until it was as far as possible removed from sand.

- Preparation of test solution: The test concentrations were set up as follows: 17.90 mg test substance were dissolved in 100 mL acetone (analytical grade) to obtain the Pre-stock solution (KV). This stock solution was stirred on a magnetic stirrer for 3 min. Several dilutions in acetone were made from this stock solution to obtain the solutions (S1 – S6) for each test concentration. The spiked sediments were placed in the vessels, 4 x 140 g sediment /test concentration and test medium M7 was added up to a final water volume of 0.38 L to produce a sediment-water volume ratio of 1:4. Detailed information are summarized within the Table 1 in section" Any other information on material and methods incl. tables"

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: non-biting midge
- Source: University of Frankfurt am Main (Germany)
- Details on collection: Transferred in July 2006 to the lab and kept since then
- Age of parental stock (mean and range, SD): 2 to 3 weeks adults were mating and laid eggs on the water surface which were used for the test
- Breeding conditions: For breeding the midges are kept in cages (40 x 40 x 40 cm), with a gauze on each side of the cage. A glass basin (diameter 19 cm and 8 cm height) is set on the bottom of each cage. The bottom of the basins are covered with a thin layer of "Kieselgur" (silica) and a 4 - 5 cm high layer of reconstituted water M7 according to Elendt, which is aerated gently. The culture conditions are 20 ± 2°C and 16 to 8 hours light-dark-cycle (light intensity ca. 500 - 1000 lux). Food: green algae and an aqueous suspension of a plant material based fish food (Tetra Phyll®)
- Handling of egg masses and larvae: The hatched larvae are fed with green algae and an aqueous suspension of a plant material based fish food (Tetra Phyll®). After 2 to 3 weeks the adults emerge. After mating, female adults will lay egg masses on the water surface where these can be taken to start a new culture or to perform toxicity tests.
- Age of animals at beginning of exposure: First instar (L1) of Chironomus riparius larvae. L1 larvae were obtained by introducing some fresh egg masses in small dishes with test medium. Two to three days after hatching the L1-larvae were transferred carefully with a blunt pipette to the test vessels.
- Feeding during test: yes
- Food type: commercial ornamental fish food extract (trade name Tetra Phyll®) as used for the breeding
- Amount: about 0.5 - 1 mg Tetraphyll® /Larvae/day per container
- Frequency: at least about three times per week

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Remarks:

The quality of the sediment is checked at least once a year (non-GLP data) for contaminants (e.g. heavy metals). The artificial sediment was prepared in the Institute for Ecotoxicology.

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

in overlaying water:
test start (0 d) 249.2 mg CaCO3/L and test end (28 d) 249.2 mg CaCO3/L (control)
test start (0 d) 267.0 mg CaCO3/L and test end (28 d) 267.0 mg CaCO3/L (80.0 μg a.s./kg dw sed)

Test temperature:

in overlaying water:
test start (0 d) 20.6 °C and test end (28 d) 20.6 °C (control)
test start (0 d) 20.8 °C and test end (28 d) 20.5 °C (solvent control)
test start (0 d) 20.8 to 20.9 °C and test end (28 d) 20.2 to 20.6 °C (test concentrations)
Total mean: 20.5 ± 0.2 °C

pH:

in overlaying water:
test start (0 d) 8.1 and test end (28 d) 8.3 to 8.4 (control)
test start (0 d) 8.2 and test end (28 d) 8.0 to 8.5 (solvent control)
test start (0 d) 8.1 to 8.2 and test end (28 d) 7.9 to 8.5 (test concentrations)

Dissolved oxygen:

in overlaying water:
test start (0 d) 8.5 mg O2/L and test end (28 d) 8.6 to 8.7 mg O2/L (control)
test start (0 d) 8.4 mg O2/L and test end (28 d) 8.6 to 8.7 mg O2/L (solvent control)
test start (0 d) 8.4 to 8.5 mg O2/L and test end (28 d) 8.2 to 8.7 mg O2/L (test concentrations)

Ammonia:

in overlaying water:
test start (0 d) 0.89 ammonium mg/L and test end (28 d) 18.6 ammonium mg/L (control)
test start (0 d) 1.16 ammonium mg/L and test end (28 d) 12.2 ammonium mg/L (80.0 μg a.s./kg dw sed)

Nominal and measured concentrations:

control, solvent control, 2.50, 5.00, 10.0, 20.0, 40.0 and 80.0 μg a.s./ kg sediment (dw) (nominal)
control, solvent control, 1.98, 3.26, 6.76, 12.73, 26.87, 59.75 μg a.s./kg dw sed. (initially measured concentrations of the test item)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 0.6 L glass beakers (Ø 9.5 cm)
- Sediment volume: The bottom of the test vessels were covered with a 1.5 cm layer of wet sediment (corresponding to about 140 g).
- Overlying water volume: Test water poured slowly into the vessels (final test water volume: 0.38 L).
- Depth of sediment and overlying water: The height of the water column was 6.0 cm.
- Aeration: yes
- Aeration frequency and intensity: Test vessels were aerated during the equilibration phase. The aeration of the water was stopped for 24 hours after insertion of test organisms and re-started 24 h later. Gentle aeration was provided through a glass Pasteur pipette situated about 2.5 cm above the sediment layer throughout the complete study over 28 days (approximately 2 bubbles per second). Test beakers were covered by clear plastic plates, preventing evaporation.
- Replacement of evaporated test water, if any: In case of a loss of water by evaporation, the test vessels were refilled with deionised water up to the mark, which indicates a volume of 0.38 L overlying water (typically once a week).

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4
- Feeding regime: feeding during the test
- Type and preparation of food: commercial ornamental fish food extract (trade name Tetra Phyll®) as used for the breeding
- Amount of food: about 0.5 - 1 mg Tetraphyll® /Larvae/day per container
- Frequency: at least about three times per week

RENEWAL OF OVERLYING WATER
- Details on volume additions: In case of a loss of water by evaporation, the test vessels were refilled with deionised water up to the mark, which indicates a volume of 0.38 L overlying water (typically once a week).

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): M7-medium was added as test water to the sediment layer. The medium was based on de-ionised water (supplied by Currenta GmbH & Co. OHG, Energie – Wasserversorgung - Leverkusen. The quality of the deionised water is checked at least twice (non-GLP data) a year for residues and contaminants).
- Alkalinity: test start (0d) 106.8 mg CaCO3/L and test end (28 d) 178.0 mg CaCO3/L (control), test start (0d) 124.6 mg CaCO3/L and test end (28d) 160.2 mg CaCO3/L (80.0 μg a.s./kg dw sed)
- Total organic carbon: <2 mg/L
- COD: < 5 mg/L
- Total solids: < 5 mg/L
- Residual chlorine < 0.01 mg/L
- Nh4+: < 0.03 mg/L
- Ag, Hg, Cd < 0.1 µg/L
- Al, As, Bor, Cr, Cu, Co, Pb, Ni, Zn: < 1 µg/L
- Fe: 1.8 µg/L
- F-: < 100 µg/L
- Pesticide residue contaminants (54 tested): all < 0.05 µg/L
- Organochlorine contaminants (25 tested): all < 0.01 µg/L

CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- % dry weight of sphagnum moss peat: 4% sphagnum moss peat (pH 3.5, air dried, originated from Erdenwerk Gregor Ziegler GmbH D-95703 Plößberg, Germany)
- Particle size distribution: ≤ 1 mm
- % sand: 75.0 % quartz sand (grain size: 91.4% of the particles were in the range of 50-200 μm, originated form Quarzwerke GmbH D-50226 Frechen, Germany)
- % silt: 1 % calcium carbonate (chemical pure, Merck)
- % clay: 20 % kaolinite (kaolinite content of 30.2 %, pH value 5.5 – 7.5, "Kaolin W", from Erbslöh / Geisenheim, Germany)
- Method of preparation (if artificial substrate): Artificial sediment was used in the test, prepared 7 days before the start of the exposure period. The formulated sand was then mixed with the suitable amounts of sediment per test concentration by feed mixer.
- Maturation of artificial substrate (if any): yes
- Amount of pore water:
control: test start (0 d) = 20.9 g; day 4 = 23.7 g; day 8 = 22.4 g; day 28 = 22.6 g;
solvent control: test start (0 d) = 22.2 g; day 4 = 25.7 g; day 8 = 25.2 g; day 28 = 26.0 g
test concentrations: test start (0 d) = 22.2 to 22.0 g; day 4 = 20.2 to 23.3 g; day 8 = 22.3 to 27.6 g; day 28 = 23.9 to 31.0 g
- Total organic carbon (%): 2.05%
- Arsen (AS): 3.2 mg/kg
- Bismut (Bi): 0.065 mg/kg
- Cadmium (Cd): 0.078 mg/kg
- Chrom (Cr): 34 mg/kg
- Kupfer (Cu): 2.5 mg/kg
- Eisen (Fe): 2700 mg/kg
- Quecksilber (Hg): 0.17 mg/kg
- Mangan (Mn): 10 mg/kg
- Nickel (Ni): 2.3 mg/kg
- Blei (Pb): 12 mg/kg
- Palladium (Pd): < 0.23 mg/kg
- Platin (Pt): < 0.023 mg/kg
- Antimon (Sb): 2.5 mg/kg
- Zinn (Sn): 2 mg/kg

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: 500-1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): visual assessment of behavioural differences compared to the control. Daily recording of the sex, time point of emergence and number of emerged midges during the period of emergence. To determine number and sex of emerged adults, the covering plates of each test container were carefully moved and the midges, which mostly stayed at the sides of the vessels, were enumerated; after identification of the sex (male midges have feathered antennae) and removed.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: no range-finding test was performed

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

5.96 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: 95% confidence limits

Remarks:

4.27 - 7.26 μg a.s./ kg dw

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

6.93 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: 95 % confidence limits

Remarks:

5.29 - 8.31 μg a.s./ kg dw

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

6.76 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

12.7 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

12 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

development rate

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

10.7 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

development rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

6.76 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

development rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

12.7 µg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

act. ingr.

Basis for effect:

development rate

Details on results:

- Total mass of test animals at beginning of test: 80
- No. of offspring produced: 1.98 µg a.s./kg dw sed.: 75 emerged; 3.26 µg a.s./kg dw sed.: 76 emerged; 6.76 µg a.s./kg dw sed.: 67 emerged; 12.73 µg a.s./kg dw sed.: 7 emerged
- No. of emerged male and female midges (per vessel and per day): Start of emergence was at day 14 for the controls and all test concentrations from 1.98 to 6.76 μg a.s./kg dw sed. 92.5 % of the inserted (n= 160) larvae maturated to adults in the pooled controls after 28 days, fulfilling the guideline requirements.
- No. of pupae failing to emerge (per vessel and per day): Throughout the study one dead pupae was observed on day 17 at test concentration of 6.76 μg a.s./ kg dw sed (corresp. to 10 μg a.s./kg nominal).
- Percent emergence per replicate and test concentration: The emergence of the test concentration of 12.73 μg a.s./kg dw sed started at day 16, and showed a statistically significant delay compared to the controls. No emergence occurred at to two highest test concentrations of 26.87 and 59.75 μg a.s./kg dw sediment. A statistically significant difference in emergence (pooled sex) was established for test concentration of 12.87 μg a.s./kg dw sed as compared to the pooled controls, resulting in a NOEC of 6.76 μg a.s./kg dw sed. At test concentrations of 26.87 and 59.75 μg a.s./kg dw sed no midges emerged.
- Mean development rate of fully emerged midges (per replicate and treatment rate; male and female midges pooled): For the development rate (pooled sex) a statistically significant difference was found for initial measured test concentrations of 12.73 μg a.s./kg dw sed as compared to the pooled controls, resulting in a NOEC of 6.76 μg a.s./kg dw sed. The EC10 value for the development rate was determined to be 10.7 μg a.s./kg dw sed.
- Sex ratio: No statistically different distribution between females and males were determined. Therefore males and females were pooled.

Results with reference substance (positive control):

- Relevant effect levels: EC50 (48 h) = 2.21 mg/L (meets the range of 0.50 mg/L – 3.6 mg/L by OECD 235).
- Limit test: no
- Dose-response test: 0.43, 0.94, 2.07, 4.55 and 10.0 mg/L.

Reported statistics and error estimates:

ECx: ECx values (e.g. x = 10, 20) and confidence intervals after 28 days were calculated by probit (or logit, weibit, etc.) analysis or in case of failure by non-parametric-methods from the appropriate parameters (endpoints), using a commercial program.
LOEC: The LOEC determinations from the appropriate parameters (endpoints) were done, using the ANOVA procedure (α = 0.05, one sided) and properly selected multiple t-tests a commercial program. In case of a limit test (comparison of control and one treatment group only) the STUDENT t-test can be used.
NOEC: The no observed effect concentration (NOEC) is the test concentration immediately below the LOEC
SOFTWARE: Calculations were carried out using Microsoft Excel® spreadsheets. All further statistical evaluations were done using the commercial program ToxRat Professional.

Analytical Results:

During the study, the measured concentrations of the test item and its metabolite were analysed in the sediment, overlying water and pore water four times, on day -2 (spiked sediment only), 0, 4, 8 and 28 in all test levels and also in the control(s). For the samples taken on day -2, the amount of the metabolite was recalculated to the test item a

nd the sum was compared to total nominal amounts of the test item added to the test system using spiked sediment to confirm the correct application of the test item.

Sediment analysis on day -2 (directly after spiking) resulted in recoveries of the test item ranging from 70.3% to 89.6% of nominal concentrations.Total recoveries of the test item plus the metabolite range from 71.9 to 89.6%. On day 0, recoveries of the test item in the sediment ranged from 63.7% to 79.2% of nominal concentrations, i.e. a deviation by more than 20% from nominal. Therefore, biological results are based on initially measured test concentrations (day 0) of the test item in the sediment of: 1.98 – 3.26 – 6.76 – 12.73 – 26.87 – 59.75 μg a.s./kg dw sed.

Chemical analyses of the sediment, overlying water and pore water show only a small partitioning of the test item from the sediment into the water phase over time (max. 12.5% were found in the overlying water and max. 1.9% in the pore water). Analyses of the sediment over time showed recoveries of the test item ranging from 63.7% to 79.2% (mean = 69.6%) of nominal for all test concentrations on day 0. On day 4, 59.6% to 76.4% (mean = 65.7%), on day 8, 53.3% to 67.6% (mean = 58.8%) and on day 28, 42.7% to 54.4% (mean = 47.4%) of nominal were found, respectively. Analyses of the overlying water over time showed recoveries of the test item of 9.6% to 11.1% (mean = 10.6%) of nominal applied amount of a.s. per test concentration on day 0. On day 4, 10.9% to 12.5% (mean = 11.5%), on day 8, 9.9% to 11.1% (mean = 10.4%) and on day 28, 0% to 5.4% (mean = 3.3%) of nominal were found, respectively. Analyses of the pore water over time showed recoveries of the test item of 1.4% to 1.9% (mean = 1.7%) of nominal concentrations on day 0 for all test concentrations. On day 4, 1.1% to 1.6% (mean = 1.4%), on day 8, 1.1% to 1.5% (mean = 1.3%) and on day 28, 0% to 0.9% (mean = 0.6%) of initial measured concentrations on day 0 were found, respectively.

Table 1: Analysesof the sediment (day -2)

test item nominal initial concentration [μg a.s./kg dw sed]	test item		metabolite	metabolite calculated to test item)	Total recovery of test item + metabolite
	measured conc. (means of two analyses each) [μg a.s./kg dw sed]	[%]	measured conc. (means of two analyses each) [μg a.s./kg dw sed]	(calc.) [μg a.s./kg dw sed]	(calc.) [μg a.s./kg dw sed]	[%]
control	<LOQ*)	-	<LOQ*)	-	<LOQ*)	-
solvent-control	<LOQ*)	-	<LOQ*)	-	<LOQ*)	-
2.50	2.17	86.8	<LOQ*)	-	2.17	86.9
5.00	4.48	89.6	<LOQ*)	-	4.48	89.7
10.0	8.57	85.7	<LOQ*)	-	8.57	85.7
20.0	16.90	84.5	<LOQ*)	-	16.90	84.5
40.0	28.13	70.3	0.61	0.63	28.76	71.9
80.0	59.87	74.8	1.48	1.53	61.40	76.7

*) The limit of quantitation (LOQ) of the method is 2 μg/kg for the test item and metabolite.

1) calculated: measured conc. of the metabolite x 544.88 (molecular weight of the test item) / 526.86 (molecular weight of the metabolite)

Table 2: Analysis of the test item (day 0, 4, 8 and 28)

Test item nominal initial concentration [μg a.s./kg dw sed] (μg a.s./tv)	day	Sediment			Overlying water			Pore water
		measured conc. of the test item (means of two analyses each)			measured conc. of the test item (means of two analyses each)			measured conc. of the test item (means of two analyses each)
		μg a.s./kg dw sed	μg a.s./tv1)	[%]4)	μg a.s./L	μg a.s./tv2)	[%]4	μg a.s./L	μg a.s./tv3	[%]4)
control	0	<LOQ*)	-	-	<LOQ*)	-	-	<LOQ*)	-	0
	4
	8
	28
solvent-control	0	<LOQ*)	-	-	<LOQ*)	-	-	<LOQ*)	-	0
	4
	8
	28
2.50 (0.243)1)	0	1.98	0.193	79.2	0.069	0.026	10.8	0.193	0.004	1.7
	4	1.49	0.145	59.6	0.073	0.028	11.3	0.124	0.003	1,1
	8	1.69	0.165	67.6	0.071	0.027	11.1	0.134	0.004	1.5
	28	1.36	0.132	54.4	0.000	0.000	0.0	0	0.000	0.0
5.00 (0.487)1)	0	3.26	0.317	65.2	0.143	0.054	11.1	0.427	0.009	1.9
	4	3.03	0.295	60.6	0.147	0.056	11.5	0.287	0.006	1.3
	8	2.81	0.274	56.2	0.127	0.048	9.9	0.239	0.005	1.1
	28	2.25	0.219	45.0	0.069	0.026	5.4	0.124	0.003	0.7
10.0 (0.973)1)	0	6.76	0.657	67.6	0.245	0.093	9.6	0.810	0.016	1.7
	4	7.64	0.743	76.4	0.301	0.114	11.7	0.589	0.014	1.4
	8	5.80	0.565	58.0	0.254	0.097	9.9	0.569	0.013	1.3
	28	4.91	0.478	49.1	0.089	0.034	3.5	0.225	0.006	0.6
20.0 (1.946)1)	0	12.73	1.239	63.7	0.523	0.199	10.2	1.83	0.038	1.9
	4	12.40	1.207	62.0	0.581	0.221	11.3	1.26	0.029	1.5
	8	10.65	1.036	53.3	0.531	0.202	10.4	0.920	0.022	1.1
	28	8.53	0.830	42.7	0.210	0.080	4.1	0.541	0.017	0.9
40.0 (3.892)1)	0	26.87	2.614	67.2	1.12	0.427	11.0	2.67	0.055	1.4
	4	28.20	2.744	70.5	1.12	0.424	10.9	2.26	0.046	1.2
	8	25.00	2.433	62.5	1.04	0.396	10.2	2.21	0.053	1.4
	28	19.91	1.938	49.8	0.356	0.135	3.5	1.39	0.033	0.9
80.0 (7.784)1)	0	59.75	5.814	74.7	2.19	0.833	10.7	6.30	0.127	1.6
	4	51.94	5.054	64.9	2.56	0.974	12.5	5.37	0.124	1.6
	8	44.23	4.304	55.3	2.21	0.839	10.8	4.77	0.114	1.5
	28	34.67	3.373	43.3	0.697	0.265	3.4	2.24	0.055	0.7

*) The limit of quantitation (LOQ) of the method for sediment is 2 μg/L for the test item and its metabolite. For water analysis the LOQ of the method is 0.0625 μg/L for the test item and 0.05 μg/L for the metabolite.

1) related to the real amount of sediment (dw) per test vessel (140 g wet weight sed. (moisture of 30.5%) /test vessel = 97.3 g

sediment (dw))

2) related to 380 mL overlying test water per test vessel

3) related to the real amount of pore water per test vessel (table 9)

4) of initial nominal / test vessel

Table 3: Analysis of the metabolite (day 0, 4, 8 and 28)

The metabolite nominal initial concentration [μg a.s./kg dw sed] (μg a.s./tv)	day	Sediment				Overlying water				Pore water
		measured conc. the metabolite (means of two analyses each)				measured conc. the metabolite (means of two analyses each)				measured conc. the metabolite (means of two analyses each)
		μg /kg	calculated4)			μg /L	calculated4)			μg /L	calculated4)
			μg /kg	μg /tv1)	[%]5)		μg /L	μg /tv2)	[%]5)		μg /L	μg /tv3)	[%]5)
control	0	<LOQ*)	0	0	0	<LOQ*)		-	-	<LOQ*)		-	0
	4
	8
	28
solvent-control	0	<LOQ*)	0	0	0	<LOQ*)		-	-	<LOQ*)		-	0
	4
	8
	28
2.50 (0.243)1)	0	<LOQ*)	0	0	0	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	4	<LOQ*)	0	0	0	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	8	<LOQ*)	0	0	0	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	28	0.71	0.73	0.071	29.2	<LOQ*)	0	0	0	<LOQ*)	0	0	0
5.00 (0.487)1)	0	<LOQ*)	0	0.0	0.0	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	4	<LOQ*)	0	0.0	0.0	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	8	0.26	0.264	0.026	5.3	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	28	2.15	2.22	0.216	44.3	<LOQ*)	0	0	0	<LOQ*)	0	0	0
10.0 (0.973)1)	0	<LOQ*)	0	0.0	0.0	<LOQ*)	0	0	0	<LOQ*)	0	0	0
	4	0.80	0.83	0.081	8.3	0.063	0.065	0.025	2.5	<LOQ*)	0	0	0
	8	1.41	1.45	0.142	14.5	0.077	0.080	0.030	3.1	<LOQ*)	0	0	0
	28	3.17	3.28	0.319	32.8	0.094	0.097	0.037	3.8	<LOQ*)	0	0	0
20.0 (1.946)1)	0	0,87	0.90	0.088	4.5	0.050	0.052	0.019	1.0	<LOQ*)	0	0	0
	4	1.34	1.39	0.135	6.9	0.124	0.128	0.049	2.5	0.058	0.060	0.001	0
	8	2.94	3.04	0.296	15.2	0.184	0.190	0.072	3.7	<LOQ*)	0	0	0
	28	7.74	8.01	0.779	40.0	0.181	0.187	0.071	3.7	0.057	0.059	0.002	0.09
40.0 (3.892)1)	0	1.46	1.51	0.147	3.8	0.113	0.117	0.044	1.1	<LOQ*)	0	0	0
	4	3.45	3.57	0.347	8.9	0.252	0.261	0.099	2.5	<LOQ*)	0	0	0
	8	4.22	4.36	0.424	10.9	0.362	0.374	0.142	3.7	<LOQ*)	0	0	0
	28	11.76	12.16	1.183	30.4	0.451	0.466	0.177	4.6	0.094	0.097	0.002	0.06
80.0 (7.784)1)	0	2.66	2.75	0.267	3.4	0.203	0.210	0.080	1.0	<LOQ*)	0	0	0
	4	6.77	7.00	0.681	8.7	0.508	0.525	0.200	2.6	<LOQ*)	0	0	0
	8	9.47	9.79	0.953	12.2	0.746	0.772	0.293	3.8	0.097	0.100	0.002	0.03
	28	34.79	35.98	3.500	45.0	0.936	0.968	0.368	4.7	0.163	0.169	0.004	0.05

*) The limit of quantitation (LOQ) of the method for sediment is 2μg/L for the test item and the metabolite.For water analysis

the LOQ of the method is 0.0625 μg/L for the test item and 0.05 μg/L for the metabolite.

1) related to the real amount of sediment (dw) per test vessel (140 g wet weight sed. (moisture of 30.5 %) /test vessel = 97.3 g

sediment (dw))

2) related to 380 mL overlying test water per test vessel

3) related to the real amount of pore water per test vessel (table 9)

4) calculated: measured conc. of the metabolite x 544.88 (molecular weight of the test item) / 526.86 (molecular weight

of the metabolite)

5) % of initial nominal (calc.)/ test vessel

Table 4: Emergence of males and females as dependent on conc. and time

Cumulative emergence of Chironomus riparius males and females as dependent on concentration (initial measured conc. in the sediment on day 0) of the test item and time (calculated from InputRawDataAll)

Treatm. [μg a.s./ kg dw]	control	solvent contr.	1.98	3.26	6.76	12.73	26.87	59.75
0 d:	0	0	0	0	0	0	0	0
10 d:	0	0	0	0	0	0	0	0
11 d:	0	0	0	0	0	0	0	0
12 d:	0	0	0	0	0	0	0	0
13 d:	0	0	0	0	0	0	0	0
14 d:	5	9	9	3	11	0	0	0
15 d:	27	29	26	16	33	0	0	0
16 d:	41	46	46	31	45	1	0	0
17 d:	56	63	57	51	57	1	0	0
18 d:	70	67	67	63	64	1	0	0
19 d:	71	68	73	71	64	1	0	0
20 d:	72	69	74	74	66	1	0	0
21 d:	73	71	75	76	67	3	0	0
22 d:	73	71	75	76	67	4	0	0
23 d:	73	71	75	76	67	4	0	0
24 d:	73	71	75	76	67	4	0	0
25 d:	73	71	75	76	67	4	0	0
26 d:	73	71	75	76	67	6	0	0
27 d:	73	71	75	76	67	6	0	0
28 d:	73	71	75	76	67	7	0	0

 

Table 5: Emergence rate of males and females of Chironomus riparius

Failure of emergence caused by the test item at 28 d.

Treatm.[μg a.s./ kg dw]	Introduced	Emerged	Not-emerged	% Not-emerged
Pooled Control	160	148	12	7.5
1.98	80	75	5	6.3
3.26	80	76	4	5.0
6.76	80	67	13	16.3
12.73	80	7	73	91.3
26.87	80	0	80	100.0
59.75	80	0	80	100.0

The control mortality of 7.5% at 28 d will be compensated using Abbott`s formula

  

Table 6: Development rate (pooled sex)

as dependent on concentration of the test item and time; Mean: arithmetic mean; Standard dev.: standard deviation; n: number of replicates; CV: coefficient of variation

Treatm.[μg a.s./ kg dw]	control	solvent-contr.	1.98	3.26	6.76	12.73
28 d	0.062	0.066	0.066	0.061	0.067	0.049
	0.063	0.066	0.060	0.058	0.067	0.046
	0.067	0.061	0.065	0.065	0.066	0.047
	0.063	0.066	0.065	0.062	0.063
Mean:	0.064	0.065	0.064	0.061	0.066	0.047
Std.Dev.:	0.0021	0.0026	0.0028	0.0026	0.0018	0.0016
n:	4	4	4	4	4	3
CV:	3.3	4.0	4.4	4.2	2.7	3.5

 

Table 7: % Inhibition of development rate of males and females after 28 d

% Inhibition of development rate of males and females caused by the test item after 28 d.

Treatm.[μg a.s./ kg dw]	Mean	Std. Dev.	N	% Inhibition
Pooled Control	0.064	0.0022	8	0.0
1.98	0.064	0.0028	4	0.3
3.26	0.061	0.0026	4	4.2
6.76	0.066	0.0018	4	-2.2
12.73	0.047	0.0016	3	26.8

*) negative inhibition values mean stimulation in the development rate

Validity criteria fulfilled:

yes

Reference 4

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Oct 2014 - 25 Jun 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 219 (Sediment-Water Chironomid Toxicity Test Using Spiked Water)

Version / remarks:

adopted 2004

Deviations:

yes

Remarks:

pore water and sediment content of the test substance were not analysed since an already existing spiked sediment study provides these information

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

yes

Details on sampling:

Water Samples
- Concentrations: test item and its metabolite: control, solvent control, 0.20; 0.40; 0.80; 1.60; 3.20 and 6.40 μg a.s./L
- Sampling interval: Samples were taken four times during the study: 1 hour, 4 days, 8 days and 28 days after application in one additional test vessel of each nominal initial test concentration; water samples (10 mL) were taken and added with 2.5 mL acetonitrile
- Sample storage before analysis: Samples were stored in a freezer until they were analysed

Vehicle:

yes

Remarks:

Dimethylformamide

Details on sediment and application:

PREPARATION OF SPIKED WATER
- Details of spiking: 17.89 mg test item was dissolved in 250 mL DMF; Stock solutions was stirred for 5 minutes and appropriate amounts of the application solution were applied into the overlying water column of the vessels as a singular event on study day 0, just below the water surface with a pipette by gently mixing of the water body ensuring homogeneous distribution of the test item without disturbance the sediment. Details are listed within the table 1 in section "Any other information on material and methods incl. table".
- Controls: yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: non-biting midge
- Source: University of Frankfurt am Main (Germany)
- Details on collection: Transferred in July 2006 to the lab and kept since then; breeding water: M7-medium, based on deionised water
- Age of parental stock (mean and range, SD): 2 to 3 weeks adults were mating, female adults will lay egg masses on the water surface which were used for the tests.
- Breeding conditions: kept in cages (40 x 40 x 40 cm), with gauze on each side of the cage. Suitable basins are set on the bottom of each cage. The bottom of the basins are covered with a thin layer of "Kieselgur" (silica) and a 4 - 5 cm high layer of reconstituted water M7 according to Elendt, which is aerated gently; culture conditions are 20 ± 2°C and 16 to 8 hours light-dark-cycle (light intensity ca. 500 - 1000 lux)
- Handling of egg masses and larvae: The hatched larvae are fed with green algae and an aqueous suspension of a plant material based fish food (Tetra Phyll®). After 2 to 3 weeks the adults emerge. After mating, female adults will lay egg masses on the water surface where these can be taken to start a new culture or to perform toxicity tests.
- Age of animals at beginning of exposure: First instar (L1) of Chironomus riparius larvae. L1 larvae were obtained by introducing some fresh egg masses in small dishes with test medium. Two to three days after hatching the L1-larvae were transferred carefully with a blunt pipette to the test vessels.
- Feeding during test: yes
- Food type: commercial ornamental fish food extract (trade name Tetra Phyll®) as used for the breeding
- Amount:about 0.5 - 1 mg Tetraphyll® /Larvae/day per container
- Frequency: at least about three times per week

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

231 to 303 mg CaCO3/L (control)
285 to 303 mg CaCO3/L (highest test item concentration)

Test temperature:

overlying water:
20.2 to 20.5 °C (control)
20.3 to 20.5 °C (solvent control)
20.0 to 20.5 °C (test concentrations)

pH:

overlying water:
8.4 to 8.6 (control)
8.4 to 8.6 (solvent control)
8.4 to 8.6 (test concentrations)

Dissolved oxygen:

overlying water:
8.2 to 8.7 mg/L; 91 to 96% (control)
8.3 to 8.7 mg/L (solvent control)
8.1 to 8.7 mg/L (test concentrations)

Ammonia:

1.16 to 14.7 ammonium mg/L (control)
1.26 to 23.3 ammonium mg/L (highest test item concentration)

Nominal and measured concentrations:

control, solvent control, 0.20; 0.40; 0.80; 1.60; 3.20 and 6.40 μg a.s./L (nominal)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 0.6 L glass beakers (Ø 9.5 cm)
- Sediment volume: 1.5 cm layer of wet sediment (corresponding to about 140 g)
- Overlying water volume: 0.38 L
- Depth of sediment and overlying water: Height of the water column was 6.0 cm
- Aeration: yes
- Aeration frequency and intensity: Aeration during the equilibration phase. Stop of aeration for 24 h after insertion of test organisms and re-started just before application of the test item. Gentle aeration was provided through a glass Pasteur pipette situated about 2.5 cm above the sediment layer throughout the complete study over 28 days (approximately 2 bubbles per second). Test beakers were covered by clear plastic plates, preventing evaporation.
- Replacement of evaporated test water, if any: In case of a loss of water by evaporation, the test vessels were refilled with deionised water up to the mark, which indicates a volume of 0.38 L overlying water (typically once a week).

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4
Feeding regime:
- Type and preparation of food: three times per week with ornamental fish food extract (trade name Tetra Phyll®)
- Amount of food: 0.5 - 1 mg Tetraphyll® /Larvae/day

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): M7-medium, based on de-ionised water (supplied by Currenta GmbH & Co. OHG, Energie – Wasserversorgung - Leverkusen). The quality of the deionised water is checked at least twice (non-GLP data) a year for residues and contaminants (e.g. pesticides and heavy metals)
- Alkalinity: 178 to 214 mg CaCO3/L (control); 178 to 249 mg CaCO3/L (control)
- Total solids: < 5 mg/L
- Total organic carbon:< 2 mg/L
- Chemical oxygen demand: < 5 mg/L
- NH4+: < 0.03 mg/L
- Residual chlorine: < 0.01 mg/L
- Inorganic contaminents: < 100 μg/L: Bor; F-; < 0.1 μg/L: Cd; Hg; Ag; < 1 μg/L: Pb; Fe; Co; Cu; Cr; As; Zn; Al ; Ni

CHARACTERIZATION OF SEDIMENT
- Equilibration period: 7 days before test start
- % dry weight of sphagnum moss peat: 4% sphagnum moss peat (pH 3.5, particel size distribution ≤ 1 mm, air dried, originated from Erdenwerk Gregor Ziegler GmbH D-95703 Plößberg, Germany)
Particle size distribution:
- % sand: 75.0% quartz sand (grain size: 91.4% of the particles were in the range of 50-200 μm, originated form Quarzwerke GmbH, Frechen, Germany)
- % silt: 1% calcium carbonate (chemical pure, Merck) to adjust the pH value to 7 ± 0.5 (figures refer to dry weight)
- % clay: 20% kaolinite (kaolinite content of 30.2%, pH value 5.5 – 7.5, "Kaolin W", from Erbslöh / Geisenheim, Germany
- Contaminants mg/kg: Arsen (As) 3.2; Bismut (Bi) 0.057; Cadmium (Cd) 0.12; Chrom (Cr) 43; Kupfer (Cu) 2.9; Eisen (Fe) 2800; Quecksilber (Hg) 0.022; Mangan (Mn) 14; Nickel (Ni) 2.7; Blei (Pb) 13; Palladium (Pd) < 0.23; Platin (Pt) < 0.023; Antimon (Sb) 2.4; Zinn (Sn) 2.1
- Other: water content 32.8%
- pH dry matter and/or whole sediment: 6.7
- Total organic carbon (%): 2.13%
- Proof of absence of chemical contaminants: The quality of the sediment is checked at least once a year (non-GLP data) for contaminants (e.g. heavy metals). The artificial sediment was prepared in the Institute for Ecotoxicology. The results of the sediment quality analyses are filed within the raw data.

OTHER TEST CONDITIONS
Light quality:
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: 500-1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The sex, time point of emergence and number of emerged midges was recorded daily during the period of emergence.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: no range-finding test was performed

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

0.71 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: pooled sex

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

1.2 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: pooled sex

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: pooled sex

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.8 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: pooled sex

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks:

pooled sex

Remarks on result:

other: 95% confidence limits

Remarks:

0.09 - 1.83 µg/L

Duration:

28 d

Dose descriptor:

other: EC20

Effect conc.:

2.01 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks:

pooled sex

Remarks on result:

other: 95% confidence limits

Remarks:

0.58 - 3.15 µg/L

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks:

pooled sex

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.8 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks:

pooled sex

Details on results:

- Total mass of test animals at beginning of test: 80
- No. of emerged male and female midges (per concentration): 150 (pooled control); 71 (0.20 µg a.s./L); 67 (0.40 µg a.s./L); 73 (0.8 µg a.s./L); 65 (1.60 µg a.s./L); 48 (3.20 µg a.s./L) and 6 (6.40 µg a.s./L)
- No. of pupae failing to emerge (per concentaion): 10 (pooled control); 9 (0.20 µg a.s./L); 13 (0.40 µg a.s./L); 7 (0.8 µg a.s./L); 15 (1.60 µg a.s./L); 32 (3.20 µg a.s./L) and 74 (6.40 µg a.s./L)
- Percent not-emergenced per test concentration: 6.3% (pooled control); 11.3% (0.20 µg a.s./L); 16.3% (0.40 µg a.s./L); 8.8% (0.8 µg a.s./L); 18.8% (1.60 µg a.s./L); 40.0% (3.20 µg a.s./L) and 92.5% (6.40 µg a.s./L)
- Mean development rate of fully emerged midges (per replicate and treatment rate; male and female midges pooled): 0.065 (control); 0.064 (solvent control); 0.067 (0.20 µg a.s./L); 0.064 (0.40 µg a.s./L); 0.066 (0.8 µg a.s./L); 0.051 (1.60 µg a.s./L); 0.042 (3.20 µg a.s./L) and 0.038 (6.40 µg a.s./L)
- Other: Analytical and biological results are summarized in Tables 1-5 within the section "Any other information on results incl. tables"

Results with reference substance (positive control):

- Dose-response test: 0.43, 0.94, 2.07, 4.55 and 10.0 mg/L
- ECx: EC50 (48 h) of 1.51 mg/L as determined in this test, meets the range defined by OECD 235 (0.50 mg/L – 3.6 mg/L)
- Other: least twice a year (i.e. event driven in case of receiving new strains, introduction of new test conditions, apparatus, etc.)

Reported statistics and error estimates:

ECx values and confidence intervals after 28 d were calculated by probit (or logit, weibit, etc.) analysis or in case of failure by non parametric-methods from the appropriate parameters (endpoints), using a commercial program. The LOEC determinations from the appropriate parameters (endpoints) were done, using the ANOVA procedure (alpha = 0.05, one sided) and properly selected multiple t-tests a commercial program. In case of a limit test (comparison of control and one treatment group only) the Student t-test can be used. Calculations were carried out using Microsoft Excel® spreadsheets. All further statistical evaluations were done using the commercial program ToxRat Professional.

For the control and solvent control the student-t-Test indicates no statistical difference for emergence rate (pooled sex) and development rate (pooled sex), therefore the two controls were pooled for further statistical analyses to increase the statistical power.

Analytical Results:

Chemical analyses of the test item and the metabolite were performed for overlying water samples over time. Analyses of the overlying water at the beginning of the exposure period (nearly one hour after spiking) reflect high recoveries of the test item with 108 % to 114 % (mean 110 %) of nominal concentrations in all test levels, thus all results and reporting are based on nominal concentrations of the test item in the overlying water, expressed in μg a.s./L. Exposure recoveries of the test item in the overlying water of all test concentrations were found after 4 days of 52% to 57% (mean 54%), after 8 days of 22% to 31% (mean 27%) and after 28 days 0% to 1.8% (mean 1.7 %). Chemical analysis of the metabolite (averages) over time yield 0.5% of nominal on day 0, 16% on day 4, 17% on day 8 and 4.4% on day 28.

Table 1: Analytical Results

	analytical results of the overlying water: average % of all nominal test concentrations
	1 hour / day 0	day 4	day 8	day 28
test item	110	54	27	1.7
metabolite	0.5	16	17	4.4

Table 2: Analysis of the test item in the overlying water

initial nominal concentration the test item [μg a.s./L]	analytical results of the test item, means of two analyses each [μg a.s./L]
	1 hour / day 0		day 4		day 8		day 28
	analysed conc.	% of initial nominal	analysed conc.	% of initial nominal	analysed conc.	% of initial nominal	analysed conc.	% of initial nominal
control	< 0.025	-	< 0.025	-	< 0.025	-	< 0.025	-
solvent-contr.	< 0.025	-	< 0.025	-	< 0.025	-	< 0.025	-
0.20	0.216	108	0.104	52	0.0442	22	< 0.025	-
0.40	0.432	108	0.217	54	0.109	27	< 0.025	-
0.80	0.872	109	0.452	57	0.219	27	< 0.025	-
1.60	1.76	110	0.826	52	0.435	27	0.0272	1.7
3.20	3.58	112	1.73	54	0.969	30	0.0520	1.6
6.40	7.30	114	3.62	57	1.96	31	0.117	1.8
average %		110		54		27		1.7

Table 3: Analysis of the metabolite in the overlying water

initial nominal concentration test item [μg a.s./L]	analytical results of the metabolite, means of two analyses each [μg a.s./L]
	1 hour / day 0		day 4		day 8		day 28
	analysed conc. calc.conc.	% of initial nominal	analysed conc. calc.conc.	% of initial nominal	analysed conc. calc.conc.	% of initial nominal	analysed conc. calc.conc.	% of initial nominal
control	< 0.025	-	< 0.025	-	< 0.025	-	< 0.025	-
solvent-contr.	< 0.025	-	< 0.025	-	< 0.025	-	< 0.025	-
0.20	< 0.025	-	0.0285 0.029	15	0.0269 0.0278	14	< 0.025 -	-
0.40	< 0.025	-	0.0537 0.056	14	0.0493 0.0510	13	< 0.025	-
0.80	< 0.025	-	0.122 0.126	16	0.104 0.108	14	0.0478 0.0494	6.2
1.60	< 0.025	-	0.277 0.286	18	0.300 0.310	19	0.102 0.105	6.6
3.20	0.0444 0.046	1.4	0.488 0.505	16	0.604 0.625	20	0.204 0.211	6.6
6.40	0.0997 0.103	1.6	1.06 1.10	17	1.35 1.40	22	0.415 0.429	6.7
average %		0.5		16		17		4.4

Calculated concentration: analysed concentration of the metabolite x 544.88 (molecular weight of the test item) / 526.86 (molecular weight of the metabolite)

Table 4: Influence on emergence and development rate after 28 days (based on nominal initial concentrations of the test item in the overlying water):

Concentrations of the test item in [μg a.s./ L]		Number of introduced larvae	Number of emerged midges	[%] Emergence of inserted larvae			Development rate (pooled sex)
Nominal	initial measured (day 0) 2)			total	male	female	(1 / d)
Controls 1)	-	160	150	93.75	45.62	48.13	0.065
0.20	0.22	80	71	88.75	48.75	40.00	0.067
0.40	0.43	80	67	83.75	45.00	38.75	0.064
0.80	0.87	80	73	91.25	46.25	45.00	0.066
1.60	1.76	80	65	81.25 3)	50.00	31.25	0.051 3)
3.20	3.58	80	48	60.00 3)	33.75	26.25	0.042 3)
6.40	7.30	80	6	7.50 3)	7.50	0	0.038 3)

1) control and solvent-control pooled

2) 1 hour after application

3) statistical significant difference (α = 0.05)

Table 5: % inhibition of development rate of males and females caused by the test item after 28 d.

Treatm.[μg a.s./L]	Mean	Std. Dev.	n	% Inhibition
Pooled Control	0.065	0.0021	8	0.0
0.20	0.067	0.0007	4	-3.2
0.40	0.064	0.0020	4	0.1
0.80	0.066	0.0022	4	-2.3
1.60	0.051	0.0035	4	20.6
3.20	0.042	0.0011	4	35.6
6.40	0.038	0.0004	3	40.9

 

Validity criteria fulfilled:

yes

Reference 5

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

30 Apr - 19 May 2015

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 (draft)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ASTM E1706 (Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrate

Version / remarks:

2005

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: Stock solutions; Control, Solvent Control, 10, 20, 40, 80, 160 and 320 µg a.i./kg (nominal)
- Sampling interval: test start (day 0) and test end (day 10)
- Sample storage before analysis: no storage

PORE WATER
- Concentrations: Stock solutions; Control, Solvent Control, 10, 20, 40, 80, 160 and 320 µg a.i./kg (nominal)
- Sampling interval: test start (day 0) and test end (day 10)
- Sample storage before analysis: no storage

OVERLYING WATER
- Concentrations: Stock solutions; Control, Solvent Control, 10, 20, 40, 80, 160 and 320 µg a.i./kg (nominal)
- Sampling interval: test start (day 0) and test end (day 10)
- Sample storage before analysis: no storage

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: The dosed sand was placed in a fume hood and the acetone was allowed to evaporate for approximately one hour. Than 70 g sand premixes were added to 630 grams of untreated formulated sediment in 2000 mL plastic Nalgene® bottles and mixed on a rotary mixer for approximately one hour. Next, 700 g of untreated formulated sediment were added to the premixes to achieve a final weight of 1400 g.
- Method of mixing: mixed on a rotary mixer for approximately 68.5 h.
- Details of spiking: A volume of 14 mL of the appropriate stock solution was added to 70.0 g of sand in a labeled glass beaker and stirred with a glass stir rod until homogenous, for each concentration.
- Controls: yes, control and solvent control
- Equilibration time: 2 d prior to the introduction of the test organisms
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 14 mL of acetone to 70.0 g of sand
- Evaporation of vehicle before use: yes, for one hour

Test organisms (species):

Chironomus dilutus (previous name: Chironomus tentans)

Details on test organisms:

TEST ORGANISM
- Common name: non-biting midge
- Source: Columbia Environmental Research Center, U.S. Geological Survey, Columbia, Missouri, USA
- Handling of egg masses and larvae: 10 midges larvae were transfered into test compartment; transfers were made below the water surface using wide-bore pipettes.
- Age of animals at beginning of exposure: Midges used in the test were second to third instar larvae, and were approximately 10-days old at test initiation
- Feeding during test: yes
- Food type: invertebrate slurry (holding period) and Tetramin® suspension of flake food in water (test)
- Amount: 1.5 mL of a 4 g/L Tetramin® suspension of flake food in water
- Frequency: daily, but not on the last days of the test

ACCLIMATION
- Acclimation period: 14 d
- Acclimation conditions (same as test or not): same conditions as for the test: temperature in the culture beakers ranged from 21.3 to 25.0 ˚C, dissolved oxygen was ≥7.5 mg/L (≥88% saturation) and the pH ranged from 8.2 to 8.7

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

10 d

Exposure phase:

total exposure duration

Hardness:

140 - 152 mg/L as CaCO3 (control)
140 - 144 mg/L as CaCO3 (solvent control)
140 - 148 mg/L as CaCO3 (255 µg a.i./kg test concentration)

Test temperature:

22.5 to 23.0 °C (control)
22.5 to 23.0 °C (solvent control)
22.6 to 22.9 °C (test concentrations)

pH:

8.1 to 8.2 (control)
8.1 to 8.2 (solvent control)
8.1 to 8.3 (test concentrations)

Dissolved oxygen:

8.2 to 8.6 mg/L (control)
8.3 to 8.6 mg/L (solvent control)
7.9 to 8.6 mg/L (test concentrations)

Ammonia:

Conductivity:

325 - 351 μS/cm (control)
329 - 367 μS/cm (solvent control)
328 - 369 μS/cm (255 µg a.i./kg test concentration)

Nominal and measured concentrations:

control, solvent control, 10, 20, 40, 80, 160 and 320 μg test item /kg based on the dry weight of the sediment (nominal)
control, solvent control, 6.6; 11; 25; 59; 127 and 255 μg a.i./kg (arithmetic mean measured concentration in sediment)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 300 mL glass beakers with stainless steel mesh screen covered holes on opposite sides of the compartment
- Sediment volume: 100 mL
- Overlying water volume: 175 mL of overlying water
- Aeration: no

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 10 (8 for the evaluation of survival and growth + 2 for analytical determination)
- No. of replicates per control / vehicle control: 10 (8 for the evaluation of survival and growth + 2 for analytical determination)
- Feeding regime: Tetramin® suspension of flake food in water (test)
- Amount: 1.5 mL of a 4 g/L Tetramin® suspension of flake food in water
- Frequency: daily, but not on the last days of the test

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): freshwater; moderately-hard water
- Source of water (if non-standard medium): from 40 meters deep located on the Wildlife International site
- Filtration: water was passed through a sand filter and pumped into a 37800 L storage tank where the water was aerated with spray nozzles. Prior to use in the test system, the water was filtered to 0.45 μm and passed through a UV sterilizer.
- Alkalinity: 178 to 182 mg/L as CaCO3 (control); 178 to 182 mg/L as CaCO3 (solvent control); 180 to 182 mg/L as CaCO3 (255 µg a.i./kg)
- Total organic carbon: 1.4%
- Pesticides and Organics (µg/L): < 0.010: Aldrin; Gamma BHC – Lindane ; Alpha BHC ; Alpha Chlordane ; Beta BHC ; Endosulfan I ; HCB Heptachlor ; Heptachlor Epoxide ; Delta BHC ; Telodrin ; < 6.3: Bolstar Coumaphos; Malathion; Demeton-O; Guthion (Azinphos-methyl); Merphos; Demeton-S; Diazinon; Methyl Parathion; Dichlorvos; Dursban (Chlorpyrifos); Disulfoton; Naled; Mevinphos; Phorate ; EPN; Ronnel; Ethion; Ethoprop; Stirophos; Ethyl Parathion; Tokuthion; Famphur; Fensulfothion; Trichloronate; Fenthion; Trithion; < 0.021 Gamma Chlordane Dieldrin; p,p-DDE; Endrin; p,p-DDT; Endrin Ketone; Endosulfan Sulfate; p,p-DDD; o,p-DDT; o,p-DDE; o,p-DDD; Mirex < 0.052; Endosulfan II < 0.031; Endrin Aldehyde < 0.10; Methoxychlor < 0.10; Kepone < 0.21; Toxaphene < 1.0; Chlordane < 0.52
- Metals: < 0.200 Aluminum; Iron; < 0.0200: Antimony; Arsenic; Zinc; Selenium; < 0.0050: Manganese; Barium; Beryllium; Vanadium; Cadmium; Silver; Cobalt; < 0.0100: Nickel;Copper; < 0.50: Nitrate Nitrogen; Nitrite Nitrogen; Fluoride; Bromide < 2.5; Potassium 7.00; Calcium 35.8; Chloride 4.0; Chromium < 0.0150; Sodium 19.1; Sulfate < 5.0; Thallium < 0.0300; Lead < 0.0150; Mercury < 0.00020; Magnesium 13.8

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5
- Particle size distribution
- % sand: 75 (industrial quartz sand)
- % silt and clay: 20
- Composition (if artificial substrate): Sandy loam with Peat Moss
- Method of preparation (if artificial substrate): dry constituents of the sediment were mixed in a PK Twinshell® mixer for 40 m and the dry soil was stored under ambient conditions until used. The pH of the sediment was 7.2.
- Moisture: 11.2% at 1/3 bar
Further constituents
- Pesticides and Organics (μg/kg): < 77: Guthion (Azinphos-methyl); Bolstar; Coumaphos; Malathion ; Demeton-O; Merphos; Demeton-S; Diazinon; Methyl Parathion; Dichlorvos; Mevinphos; Disulfoton; Naled; Dursban (Chlorpyrifos); Phorate; EPN; Ronnel; Ethion; Stirophos; Ethoprop; Famphur; Fenthion; Ethyl Parathion; Tokuthion; Trichloronate; Trithion; < 1.9: o,p-DDD o,p-DDE; Endosulfan II; o,p-DDT; Endosulfan Sulfate; p,p-DDD; Endrin; p,p-DDE; Endrin Aldehyde; p,p-DDT; Dieldrin; Mirex; < 0.95: Aldrin Gamma BHC – Lindane; Alpha BHC; Gamma Chlordane; Alpha Chlordane; HCB; Heptachlor; Endosulfan I; Heptachlor Epoxide;Methoxychlor < 7.7; Endrin Ketone < 2.1; Telodrin < 1.4; Toxaphene < 38; Fensulfothion < 230; Beta BHC < 1.1; Chlordane < 19; Kepone < 8.0; Delta BHC < 1.0
- Metals (mg/kg):Aluminum 6,170; Magnesium 2,840; Mercury < 22.0; Manganese 3.81; < 2.20: Antimony; Arsenic; Selenium; Barium 92.6; Nickel 2.81; < 0.551: Beryllium Cadmium; Cobalt; Silver; Nitrate by IC (solid) 4.6; Bromide by IC (solid) < 5.8; Nitrite by IC (solid) < 1.2; Fluoride by IC (solid) < 1.2; Potassium 80.0; Calcium 5,090; Chloride by IC (solid) 22.7; Chromium 6.79; Sodium < 110; Sulfate by IC (solid) 161; Copper < 1.10; Thallium < 3.30; Vanadium 2.63; Iron 464; Zinc 3.86; Lead 9.57
- Organic matter: 2.4%
- Sediment sieved: yes (on Day 10 of the test, the midges were removed from the sediment using a 425 μm sieve)
- pH dry matter and/or whole sediment: 7.2
- Total organic carbon (%): 1.4%
- Bulk density: 1.10 gm/cc
- CEC: 5.4 meq/100 g

OTHER TEST CONDITIONS
- Light quality: Fluorescent light bulbs (emit wavelengths similar to natural sunlight)
- Photoperiod: Photoperiod of 16 h of light and 8 h of darkness with a 30 min transition period of low light intensity
- Light intensity: 596 lux at the surface of the water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The test compartments were observed daily to make visual assessments of abnormal behavior (e.g. leaving the sediment, climbing the walls of the test compartment, swimming in the water column). On Day 10 of the test, the midges were removed from the sediment using a 425 μm sieve, and the numbers of live or dead organisms were enumerated. If the total number of individuals found in a replicate at test termination was fewer than the number initially placed into the replicate at the beginning of the test, then those missing were considered dead. Any immobile organisms removed from the sediment were also considered dead. Surviving larvae were kept separated by replicate for the determination of the average individual ash-free dry weight (AFDW) per replicate. When pupae were recovered, they were included in the survival data, but were not included in the growth data.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Results used to determine the conditions for the definitive study: test concentrations of the definitive test were based on the results of a range-finding test

Reference substance (positive control):

no

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

34 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95 % confidence limits

Remarks:

11 - 59 µg a.i./kg based on the mean measured concentration in the sediment

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

25 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

based on the mean measured concentration in the sediment

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

11 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

based on the mean measured concentration in the sediment

Details on results:

- Survial of test animals at end of exposure period (%): negative control, solvent control, 6.6, 11, 25, 59, 127 and 255 μg a.i./kg treatment groups survival was 91, 90, 89, 89, 61, 29, 0 and 1%, respectively
- Total mass of test animals at beginning of test (mean): 0.295 mg
- Changes in body weigth of live adults (% of initial weight) at end of exposure period (average): individual ash-free dry weight per midge: negative control was 1.95 mg; solvent control was 2.01 mg; 6.6 μg a.i./kg was 1.86 mg; 11 μg a.i./kg was 1.89 mg; 25 μg a.i./kg was 2.18 mg and 59 μg a.i./kg was 1.51 mg
- Other: Detailed analytical and biological results are summarized within the tables 1-7 in section "Any other information on results incl. tables"

Reported statistics and error estimates:

Since a solvent control group was used in addition to a negative control group, the data from these two groups were compared using a t-test. There were no significant differences therefore the treatment groups were compared to the pooled control. Using the statistical program TOXSTAT Version 3.5, the survival and growth (ash-free dry weight) data were evaluated (α = 0.01) for normality (Chi-Square) and homogeneity of variances (Levene’s). The survival and growth data were deemed normal with homogenous variance; therefore a Bonferroni t-test was used to identify treatment levels that were statistically different from the pooled control group.
An LC50 value was calculated for the survival/mortality data using the computer software of C.E. Stephan. The Stephan program was designed to calculate the LC50 value by non-linear interpolation and the 95% confidence limits were determined by binomial probability. The no-observed-effect-concentration (NOEC) and the lowest-observed-effect-concentration (LOEC) were determined by visual interpretation of the dose-response pattern and statistical analyses of the survival and growth (ash-free dry weight) data.

Analytical results:

Table 1: Measured Concentrations of the test item in Sediment Samples from a Sediment Toxicity Test with Chironomus dilutus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-255-)	Sampling Interval (Day)	Measured Concentration (μg a.i./kg)1,3	Percent of Nominal2,3	Mean Measured Concentration (μg a.i./kg)	Mean Percent of Nominal
0.0	S-1	0	< LOQ	--	--	--
(Negative Control)	S-9	10	< LOQ	--
0.0	S-2	0	< LOQ	--	--	--
(Solvent Control)	S-10	10	< LOQ	--
10	S-3	0	7.02	70.2	6.6	66
	S-11	10	6.27	62.7
20	S-4	0	12.8	63.9	11	55
	S-12	10	9.94	49.7
40	S-5	0	29.3	73.2	25	63
	S-13	10	20.6	51.5
80	S-6	0	60.9	76.1	59	74
	S-14	10	56.1	70.1
160	S-7	0	136	85.2	127	79
	S-15	10	117	73.1
320	S-8	0	276	86.4	255	80
	S-16	10	233	72.9

1 The limit of quantitation (LOQ) was 5.04 μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.200 μg a.i./L) and the dilution factor of the matrix blank samples (20.0), divided by the soil content (0.793) of the negative control from day 0.

2 Results were generated using Excel 2010 in full precision mode and Analyst 1.6. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

Table 2: Measured Concentrations of the test item in Overlying Water Samples from a Sediment Toxicity Test with Chironomus dilutus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-255-)	Sampling Interval (Day)	Measured the test item Concentration (μg a.i./L)1,2
0.0	OW-1	0	< LOQ
(Negative Control)	OW-9	10	< LOQ
0.0	OW-2	0	< LOQ
(Solvent Control)	OW-10	10	< LOQ
10	OW-3	0	< LOQ
	OW-11	10	< LOQ
20	OW-4	0	< LOQ
	OW-12	10	< LOQ
40	OW-5	0	< LOQ
	OW-13	10	< LOQ
80	OW-6	0	< LOQ
	OW-14	10	< LOQ
160	OW-7	0	0.540
	OW-15	10	< LOQ
320	OW-8	0	1.01
	OW-16	10	< LOQ

1 The limit of quantitation (LOQ) was 0.400 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.200 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2 Results were generated using Analyst version 1.6. Manual calculations may differ slightly.

Table 3: Measured Concentrations of the test item in Pore Water Samples from a Sediment Toxicity Test with Chironomus dilutus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-255-)	Sampling Interval (Day)	Measured test item Concentration (μg a.i./L)1,2	Mean Measured Concentration (μg a.i./L)
0.0	PW-1	0	< LOQ	< LOQ
(Negative Control)	PW-9	10	< LOQ
0.0	PW-2	0	< LOQ	< LOQ
(Solvent Control)	PW-10	10	< LOQ
10	PW-3	0	1.04	0.85
	PW-11	10	0.664
20	PW-4	0	2.02	1.7
	PW-12	10	1.41
40	PW-5**	0	6.12	4.7
	PW-13	10	3.32
80	PW-6**	0	14.5	11
	PW-14	10	7.24
160	PW-7**	0	30.6	23
	PW-15	10	15.4
320	PW-8**	0	62.2	48
	PW-16	10	32.8

1 The limit of quantitation (LOQ) was 0.400 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.200 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2 Results were generated using Analyst version 1.6. Manual calculations may differ slightly.

* Extrapolated value. **Sample reanalyzed

Table 4: Measured Concentrations of the metabolite in Sediment Samples from a Sediment Toxicity Test with Chironomus dilutus

Nominal test item Concentration (μg a.i./kg)	Sample Number (149A-255-)	Sampling Interval (Day)	Measured metabolite Concentration (μg a.i./kg)1,3	Percent of Nominal2,3
0.0	S-1M	0	< LOQ	--
(Negative Control)	S-9M	10	< LOQ	--
0.0	S-2M	0	< LOQ	--
(Solvent Control)	S-10M	10	< LOQ	--
10	S-3M	0	< LOQ	--
	S-11M	10	< LOQ	--
20	S-4M	0	< LOQ	--
	S-12M	10	< LOQ	--
40	S-5M	0	1.71	--
	S-13M	10	2.25	--
80	S-6M	0	2.60	--
	S-14M	10	5.81	--
160	S-7M	0	5.15	--
	S-15M	10	13.5	--
320	S-8M	0	13.0	--
	S-16M	10	28.4*	--

1 The limit of quantitation (LOQ) was 1.26 μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.0500 μg a.i./L) and the dilution factor of the control samples (20.0), divided by the soil content (0.793) of the negative control sediment on Day 0.

2 Results were generated using Excel 2010 in full precision mode and Analyst 1.4.1. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

* Extrapolated value.

Table 5: Measured Concentrations of the metabolite in Overlying Water Samples from a Sediment Toxicity Test with Chironomus dilutus

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-255-)	Sampling Interval (Day)	Measured metabolite Concentration (μg a.i./L)1,2
0.0	OW-1M	0	< LOQ
(Negative Control)	OW-9M	10	< LOQ
0.0	OW-2M	0	< LOQ
(Solvent Control)	OW-10M	10	< LOQ
10	OW-3M	0	< LOQ
	OW-11M	10	< LOQ
20	OW-4M	0	< LOQ
	OW-12M	10	< LOQ
40	OW-5M	0	< LOQ
	OW-13M	10	< LOQ
80	OW-6M	0	< LOQ
	OW-14M	10	< LOQ
160	OW-7M	0	< LOQ
	OW-15M	10	< LOQ
320	OW-8M	0	< LOQ
	OW-16M	10	< LOQ

1 The limit of quantitation (LOQ) was 0.100 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0500 μg a.i./L) and the dilution factor of the matrix control samples (2.00).

2 Results were generated using Analyst version 1.4.1. Manual calculations may differ slightly.

* Extrapolated value.

Table 6: Measured Concentrations of the metabolite in Pore Water Samples from a Sediment Toxicity Test with Chironomus dilutes

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-255-)	Sampling Interval (Day)	Measured metabolite Concentration (μg a.i./L)1,2
0.0	PW-1M	0	< LOQ
(Negative Control)	PW-9M	10	< LOQ
0.0	PW-2M	0	< LOQ
(Solvent Control)	PW-10M	10	< LOQ
10	PW-3M	0	< LOQ
	PW-11M	10	< LOQ
20	PW-4M	0	< LOQ
	PW-12M	10	< LOQ
40	PW-5M	0	< LOQ
	PW-13M	10	< LOQ
80	PW-6M	0	0.0763*
	PW-14M	10	0.0820*
160	PW-7M	0	0.153
	PW-15M	10	0.127
320	PW-8M	0	0.331
	PW-16M	10	0.295

1 The limit of quantitation (LOQ) was 0.100 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0500 μg a.i./L) and the dilution factor of the control samples (2.00).

2 Results were generated using Analyst version 1.6. Manual calculations may differ slightly.

* Extrapolated value.

Biological results :

Table 7: Mean Survival and Growth of the Midge (Chironomus dilutus) During a 10-Day Sediment Toxicity Test

Mean Measured Concentration in Sediment (mg a.i./kg)	Percent Survival (%)	Mean Number of Surviving Midges (± St. Dev.)1	Percent Reduction 2 (%)	Mean Individual Dry Weight (mg) (± St. Dev.)	Percent Reduction 2 (%)
Negative Control	91	9.1 (± 0.83)	--	1.95 (± 0.21)	--
Solvent Control	90	9.0 (± 1.2)	--	2.01 (± 0.18)	--
Pooled Control	91	9.1 (± 1.0)	--	1.98 (± 0.19)	--
6.6	89	8.9 (± 0.99)	2.1	1.86 (± 0.15)	6.1
11	89	8.9 (± 0.99)	2.1	1.89 (± 0.26)	4.8
25 3	61*	6.1 (± 2.1)	32	2.18 (± 0.45)	-10
59 3	29*	2.9 (± 1.6)	68	1.51 (± 0.62)	24
127 3	0*	0.0 (± 0.00)	100	0.00 (± 0.00)	100
255 3	1.3*	0.13 (± 0.35)	99	0.0204	99

1 Each replicate contained 10 midges at test initiation.

2 Percent reduction was calculated in relation to the mean of the pooled control using Excel in full precision mode, manual calculations may differ slightly.

3 The 25, 59, 127, and 255 μg a.i./kg treatment groups were excluded from the analysis of growth measurements because there were significant effects on survival at these test levels.

4 A standard deviation could not be calculated, since only one organism survived.

* There were statistically significant differences (p<0.05) in the mean number of surviving midges and the mean individual ash-free dry weight in comparison to the pooled control group using a Bonferroni t-test.

Validity criteria fulfilled:

yes

Reference 6

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 Dec 2015 - 3 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ASTM E1706 (Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrate

Version / remarks:

2005

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA 600/R-99/064

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP Number 850.1770

Version / remarks:

draft version

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: each test concentration and control and stock solutions
- Sampling interval: on Days 0, 14 and 28
- Sample storage before analysis: no storage

PORE WATER
- Concentrations: each test concentration and control and stock solutions
- Sampling interval: on Days 0, 14 and 28
- Sample storage before analysis: no storage

OVERLYING WATER
- Concentrations: each test concentration and control and stock solutions
- Sampling interval: on Days 0, 14 and 28
- Sample storage before analysis: no storage

Vehicle:

yes

Remarks:

Acetone

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: Stock solutions were prepared in acetone and further dilutions were prepared by proportional dilution with acetone, appropriate aliquots were used dosing the sand which was placed in a fume hood to allow the acetone to evaporate for approximately two hour. Than 42.5 g sand premixes were added to 958.8 g of untreated formulated sediment in plastic Nalgene® bottles and mixed on a rotary mixer for approximately two hours. Next, 941.2 g of untreated formulated sediment were added.
- Method of mixing: mixed on a rotary mixer for approximately 44.5 h.
- Details of spiking: A volume of 8.5 mL of the appropriate stock solution was added to 42.5 g of sand in a glass beaker and stirred with a glass stir rod.
- Controls: yes, control and solvent control
- Equilibration time: 2 d prior to the introduction of the test organisms
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evaporation of vehicle before use: yes, for two hours under the fume hood

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM
- Strain/clone: Hyalella azteca
- Source: original culture from Aquatic Bio Systems, Fort Collins, Colorado, USA
- Age of parental stock (mean and range, SD): Known age organisms were obtained from the culture by isolating adults in the culture 8 days prior to the start of the test; 7 days prior to the test, <24 hour old neonates were isolated from the culture tanks containing the isolated adults and held until the start of the test
- Breeding conditions: organisms were held in water from the same source as was used during testing
- Age of animals at beginning of exposure: 7 - 8 days
Feeding during test
- Food type: suspension of a yeast, cereal grass and trout chow (YCT) and suspension of TetraMin® flake food
- Amount: 1 mL
- Frequency: once daily

ACCLIMATION
- Acclimation period: 2 d
- Acclimation conditions (same as test or not): same
- Type and amount of food: TetraMin® flake food
- Feeding frequency: two to three times per week
- Health during acclimation: no sign of disease, abnormalities or sickness observed in the culture prior to the test

Study type:

laboratory study

Test type:

flow-through

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

42 d

Exposure phase:

total exposure duration

Remarks:

28 d sediment/overlying water exposure and 14 d water only exposure for monitoring reproduction

Post exposure observation period:

On Day 28, surviving amphipods were removed from the sediment and were transferred to corresponding clean, water-only test compartments and monitored for survival, reproduction and growth after Day 35 and 42.

Hardness:

Overlying water:
139 ± 2.3 mg/L as CaCO3 (Control)
135 ± 6.1 mg/L as CaCO3 (Solvent control)
133 - 137 ± 2.3 - 4.6 mg/L as CaCO3 (test item concentrations)

Test temperature:

Overlying water:
23.1 °C ± 0.18 °C (Control)
23.1 °C ± 0.17 °C (Solvent Control)
23.1 °C ± (0.15 - 0.16 °C) (test item concentrations)

pH:

Overlying water:
8.1 ± 0.07 (Control)
8.1 ± 0.10 (Solvent Control)
8.1 ± (0.07 - 0.10) (test item concetrations)
Sediment:
7.2 ± 0.12 (Control)
7.1 ± 0.10 (Solvent Control)
7.1 ± (0.00 - 0.10) (test item concetrations)
Pore water:
7.1 ± 0.52 (Control)
7.2 ± 0.53 (Solvent Control)
7.2 -7.3 ± (0.44 - 0.61) (test item concetrations)

Dissolved oxygen:

Overlying water:
8.3 ± 0.22 mg/L (Control)
8.3 ± 0.27 mg/L (Solvent control)
8.2 - 8.3 mg/L ± 0.25 - 0.27 mg/L (test item concentrations)

Ammonia:

Overlying water:
0.181 ± 0.17 mg/L as NH3 (Control)
Pore water:
6.22 ± 1.43 mg/L as NH3 (Control)
7.62 ± 1.91 mg/L as NH3 (Solvent control)
5.99 - 7.06 ± (0.96 - 2.78) mg/L as NH3 (test item concetrations)

Conductivity:

Overlying water:
383 ± 25 μS/cm (Control)
390 ± 26 μS/cm (Solvent control)
392 - 396 ± 24 - 26 μS/cm (test item concentrations)

Nominal and measured concentrations:

Control, Solvent control, 80, 160, 320, 640 and 1280 μg a.i./kg dry weight (nominal)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 300 mL glass beakers with stainless steel mesh screen covered holes on opposite sides of the compartment
- Sediment volume: 100 mL
- Overlying water volume: 175 mL overlaying water
- Depth of sediment and overlying water: sediment depth 3.0 cm in a representative test compartment and depth of overlying water 6.0 cm in a representative test compartment; from Day 28 until test end each remaining compartment contained only dilution water and no sediment. The water level in the beakers was maintained by the water levels in the stainless steel tanks (test chambers) and the position of the holes on the opposite sides of the beakers (test compartments).
- Aeration: no

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 12 (additional replicates without amphipods: 1 for water quality measurements and sediment pH measurements, and 3 for analytical sampling)
- No. of replicates per control / vehicle control: 12 (see above)
- Feeding regime: daily
- Type and preparation of food: Tetramin® suspension of flake food in water (test)
- Amount of food: see section " Any other information on material and methods incl tables"

RENEWAL OF OVERLYING WATER
- Details on volume additions: dilution additions delivered directly into the test compartments; dilution water delivery volume was verified prior to test initiation; using a time-programmed water delivery system
- Flow-rate: two volume additions of dilution water per day and replicate

OVERLYING WATER CHARACTERISTCS
- Type of water: freshwater, moderately-hard water
- Source of water: well water
- Location: Wildlife International site
- Description of sampling site: 40 meters deep
- Filtration: sand filter to remove particles greater than 25 µm, pumped into 37,800-L storage tank and aerated with spray nozzles and was again filtered (0.45 μm) and passed through a U.V. sterilizer
- Alkalinity: 179 mg/L as CaCO3 (mean) range: 176 - 180 mg/L as CaCO3
- Conductivity: 333 μS/cm (mean), range: 309 - 365 μS/cm
- Hardness: 139 mg/L as CaCO3 (mean); range 136 - 144 mg/L as CaCO3
- Total organic carbon: < 2 mg/L
- pH: 8.0 (mean), range: 7.9 - 8.1
- Pesticides and Organics (µg/L): < 0.010: Aldrin; Gamma BHC – Lindane ; Alpha BHC ; Alpha Chlordane ; Beta BHC ; Endosulfan I ; HCB Heptachlor ; Heptachlor Epoxide ; Delta BHC ; Telodrin ; < 6.3: Bolstar Coumaphos; Malathion; Demeton-O; Guthion (Azinphos-methyl); Merphos; Demeton-S; Diazinon; Methyl Parathion; Dichlorvos; Dursban (Chlorpyrifos); Disulfoton; Naled; Mevinphos; Phorate ; EPN; Ronnel; Ethion; Ethoprop; Stirophos; Ethyl Parathion; Tokuthion; Famphur; Fensulfothion; Trichloronate; Fenthion; Trithion; < 0.021 Gamma Chlordane Dieldrin; p,p-DDE; Endrin; p,p-DDT; Endrin Ketone; Endosulfan Sulfate; p,p-DDD; o,p-DDT; o,p-DDE; o,p-DDD; Mirex < 0.052; Endosulfan II < 0.031; Endrin Aldehyde < 0.10; Methoxychlor < 0.10; Kepone < 0.21; Toxaphene < 1.0; Chlordane < 0.52
- Metals: < 0.200 Aluminum; Iron; < 0.0200: Antimony; Arsenic; Zinc; Selenium; < 0.0050: Manganese; Barium; Beryllium; Vanadium; Cadmium; Silver; Cobalt; < 0.0100: Nickel;Copper; < 0.50: Nitrate Nitrogen; Nitrite Nitrogen; Fluoride; Bromide < 2.5; Potassium 7.00; Calcium 35.8; Chloride 4.0; Chromium < 0.0150; Sodium 19.1; Sulfate < 5.0; Thallium < 0.0300; Lead < 0.0150; Mercury < 0.00020; Magnesium 13.8

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Natural freshwater sediment collected at West Bearskin Lake, Minnesota, USA

HANDLING OF NATURAL SEDIMENT
- Storage conditions: stored frozen and under refrigeration after receipt in the laboratory

CHARACTERIZATION OF SEDIMENT
- Particle size distribution
- % sand: 45
- % silt: 52
- % clay: 3
- Composition (if artificial substrate): Silt loam with Peat Moss
- Moisture: 51.0% at 1/3 bar
Further constituents
- Pesticides and Organics (μg/kg): < 2.2 Aldrin, Gamma BHC – Lindane, Alpha BHC, Gamma Chlordane, Alpha Chlordane, Endosulfan I, Heptachlor, Heptachlor Epoxide; < 4.5 Dieldrin Mirex, o,p-DDD ,o,p-DDE ,Endosulfan II , o,p-DDT ,Endosulfan Sulfate , p,p-DDD ,Endrin , p,p-DDE, Endrin Aldehyde , p,p-DDT; < 180 Guthion (Azinphos-methyl) , Coumaphos , Bolstar , Malathion Demeton-O , Merphos , Demeton-S , Diazinon , Methyl Parathion, Dichlorvos , Mevinphos , Disulfoton , Naled , Dursban (Chlorpyrifos) , Phorate , EPN , Ronnel , Ethion , tirophos, Ethoprop , Famphur , Ethyl Parathion , Tokuthion , Trichloronate , Fenthion , Trithion; < 2.4 Delta BHC, < 2.7 Beta BHC, < 6.1 HCB, < 18 Methoxychlor, < 45 Chlordane, < 19 Kepone, < 4.8 Endrin Ketone, < .2 Telodrin, < 88 Toxaphene, < 530 Fensulfothion
- Metals (mg/kg):(mg/L) Aluminum 13,300, Magnesium 5,420, Antimony < 5.20, Manganese 473, Arsenic < 6.35, Mercury < 50.0, Barium 71.3, Nickel 41.6, Beryllium < 1.30, Nitrate by IC (solid) < 4.0, Bromide by IC (solid) < 13.2, Nitrite by IC (solid) < 2.6, Cadmium < 1.30, Potassium 1,620, Calcium 4,720, Selenium 7.62, Chloride by IC (solid) < 26.5, Silver < 1.30, Chromium 34.0, Sodium 478, Cobalt 12.4 , Sulfate by IC (solid) 632, Copper 59.3 , Thallium < 7.80, Fluoride by IC (solid) < 2.6 , Vanadium 58.1, Iron 49,500 , Zinc 114, Lead 6.58
- Organic matter: 14.6%
- pH dry matter and/or whole sediment: 5.3 (in soil:water ratio 1:1)
- Total organic carbon (%): 8.5%
- Bulk density: 0.71 gm/cc
- CEC: 12.5 meq/100 g

OTHER TEST CONDITIONS
- Light quality: fluorescent tubes that emitted wavelengths similar to natural sunlight
- Photoperiod: 16 hours of light and 8 hours of darkness; with a 30-minute transition period of low light intensity
- Light intensity: 572 lux

EFFECT PARAMETERS MEASURED: survival, growth and reproduction

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: a non-GLP trial was conducted to determine the appropriate acclimation period for the test substance in the water/sediment systems
- Test concentrations: 2.5 and 80 mg/kg
- Results used to determine the conditions for the definitive study: based on the results of the non-GLP pre-test the appropriate acclimation period was conducted

Reference substance (positive control):

no

Duration:

42 d

Dose descriptor:

LC50

Effect conc.:

> 992 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

42 d

Dose descriptor:

LOEC

Effect conc.:

> 992 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

>= 992 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

- Other: See section "Any other information on results incl tables"

Results with reference substance (positive control):

No reference substance was used.

Reported statistics and error estimates:

All statistical analyses were performed using TOXSTAT version 3.5 or SAS version 9.4 (7, 8). Since a solvent control was included in the test design, the data from the negative and solvent control groups were compared to determine if there were any statistical differences. Survival data was evaluated (α = 0.01) for normality (Chi-Square) and homogeneity of variances (Levene’s Test). The length and weight data were deemed normal (Shapiro-Wilk’s) with homogenous variance (Levene’s Test); differences between the treatment groups and the controls were detected via Dunnett’s test (one tailed, α = 0.05). The reproduction data were evaluated for normality and homogeneity of variances. The reproduction data was deemed normal (Shapiro-Wilk’s) with homogenous variance (Levene’s Test); therefore, a Dunnett’s test was used to identify differences between the treatment groups and the pooled control group (one tailed, α = 0.05).

Table 1: Analytical Results: Measured Concentrations of the test item in Dosing Stock Solution Samples

Nominal Concentration (μg a.i./mL)	Sample Number (149A-259-)	Measured Concentration (μg a.i./mL)	Percent of Nominal 1)
8	St1	8.48	106
16	St2	16.7	104
32	St3	33.1	103
64	St4	66.2	103
128	St5	131	102

1) Results were generated using OpenLab version A.01.05. Manual calculations may differ slightly.

Table 2: Analytical Results: Measured Concentrations of the test item in Sediment Samples from a Sediment Toxicity Test with Hyalella azteca

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-259-)	Sampling Time (Days)	Measured Concentration (μg a.i./kg)1,2,3	Percent of Nominal2	Mean Measured Concentration (μg a.i./kg)	Mean Measured Percent of Nominal
Negative Control	1-SED	0	< LOQ	--	--	--
	8-SED*	14	< LOQ	--	--	--
	15-SED	28	< LOQ	--	--	--
Solvent Control	2-SED	0	< LOQ	--	--	--
	9-SED*	14	< LOQ	--	--	--
	16-SED*	28	<LOQ	--	--	--
80	3-SED	0	75.3	94.1	58 ± 19.7	73
	10-SED	14	61.6	77.0	CV=34.0	--
	17-SED	28	36.4	45.5	--	--
160	4-SED	0	141	88.2	109 ± 37.0	68
	11-SED	14	118	73.8	CV = 33.9	--
	18-SED	28	68.5	42.8	--	--
320	5-SED	0	295	92.1	239 ± 65.6	75
	12-SED	14	256	80.0	CV = 27.4	--
	19-SED	28	167	52.1	--	--
641	6-SED	0	588	91.9	475 ± 128	74
	13-SED	14	501	78.3	CV = 26.9	--
	20-SED	28	336	52.5	--	--
1280	7-SED	0	1209	94.4	992 ± 263	78
	14-SED	14	1066	83.3	CV = 26.5	--
	21-SED	28	700	54.7	--	--

1 The limit of quantitation (LOQ) was 17.4 μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.200 μg a.i./L) and the dilution factor (40.0) of the matrix blank samples from day 14, divided by the soil content (0.461) of the negative control sample from day 14.

2 Results were generated using Analyst versions 1.4.1, 1.6.2 and Excel 2010 in the full precision mode. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

* Sample reanalyzed.

Table 3: Analytical Results: Measured Concentrations of the main metabolite in Sediment Samples from a Sediment Toxicity Test with Hyalella azteca

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-259-)	Sampling Time (Days)	Measured Concentration (μg a.i./kg) 1,2,3
Negative Control	M1-SED	0	< LOQ
(0.0)	M8-SED	14	< LOQ
	M15-SED	28	< LOQ
Solvent Control	M2-SED*	0	< LOQ
(0.0)	M9-SED	14	< LOQ
	M16-SED	28	< LOQ
80	M3-SED*	0	< LOQ
	M10-SED	14	7.27
	M17-SED	28	9.37
160	M4-SED	0	< LOQ
	M11-SED	14	13.1
	M18-SED	28	18.8
320	M5-SED	0	< LOQ
	M12-SED	14	30.4
	M19-SED	28	37.8
641	M6-SED	0	< LOQ
	M13-SED	14	69.7
	M20-SED	28	84.5**
1280	M7-SED	0	6.25
	M14-SED*	14	143
	M21-SED*	28	210

1 The limit of quantitation (LOQ) was 3.30 μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.100 μg a.i./L) and the dilution factor of the negative control sample (20.0) divided by the soil content (0.606) of the negative control sample on day 0.

2 Results were generated using Analyst versions 1.4.1, 1.6.2 and Excel 2010 in the full precision mode. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

* Sample reanalyzed.

**Extrapolated value.

Table 4: Analytical Results: Measured Concentrations of the test item in Overlying Water Samples from a Sediment Toxicity Test with Hyalella Azteca

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-259-)	Sampling Time (Days)	Measured Concentration (μg a.i./L)1,2
Negative Control	1-OW	0	<LOQ
(0.0)	8-OW	14	< LOQ
	15-OW	28	< LOQ
Solvent Control	2-OW	0	< LOQ
(0.0)	9-OW	14	< LOQ
	16-OW	28	< LOQ
80	3-OW	0	< LOQ
	10-OW	14	< LOQ
	17-OW	28	< LOQ
160	4-OW	0	< LOQ
	11-OW	14	< LOQ
	18-OW	28	< LOQ
320	5-OW	0	0.657
	12-OW	14	< LOQ
	19-OW	28	< LOQ
641	6-OW	0	1.40
	13-OW	14	0.583
	20-OW	28	< LOQ
1280	7-OW	0	2.47
	14-OW	14	0.816
	21-OW	28	< LOQ

1 The limit of quantitation (LOQ) was 0.400 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.200 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2 Results were generated using Analyst versions 1.4.1 and 1.6.2. Manual calculations may differ slightly.

Measured Concentrations of BCS-CQ63359 in Overlying Water Samples from a Sediment Toxicity Test with Hyalella azteca, all values were < LOQ (the limit of quantitation was 0.200 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.100 μg a.i./L) and the dilution factor of the negative control samples (2.00))

Table 5: Analytical Results: Measured Concentrations of the test item in Pore Water Samples from a Sediment Toxicity Test with Hyalella Azteca

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-259-)	Sampling Time (Days)	Measured Concentration (μg a.i./L)1,2
Negative Control	1-PW	0	< LOQ
(0.0)	8-PW	14	< LOQ
	15-PW	28	< LOQ
Solvent Control	2-PW	0	< LOQ
(0.0)	9-PW	14	< LOQ
	16-PW	28	< LOQ
80	3-PW	0	3.38
	10-PW	14	2.47
	17-PW	28	2.11
160	4-PW*	0	7.33
	11-PW	14	5.02
	18-PW	28	4.17**
320	5-PW*	0	15.2
	12-PW	14	10.7
	19-PW	28	9.08
641	6-PW*	0	32.3
	13-PW	14	22.7
	20-PW	28	18.8
1280	7-PW*	0	67.5
	14-PW	14	47.5
	21-PW	28	35.0

1 The limit of quantitation (LOQ) was 0.400 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.200 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2 Results were generated using Analyst versions 1.4.1 and 1.6.2. Manual calculations may differ slightly.

* Samples reanalyzed. **Extrapolated value.

Table 6: Analytical Results: Measured Concentrations of the main metabolite in Pore Water Samples from a Sediment Toxicity Test with Hyalella azteca

Nominal Test Concentration (μg a.i./kg)	Sample Number (149A-259-)	Sampling Time (Days)	Measured Concentration (μg a.i./L)1,2
Negative Control	M1-PW	0	< LOQ
(0.0)	M8-PW	14	< LOQ
	M15-PW	28	< LOQ
Solvent Control	M2-PW	0	< LOQ
(0.0)	M9-PW	14	< LOQ
	M16-PW	28	< LOQ
80	M3-PW	0	< LOQ
	M10-PW	14	< LOQ
	M17-PW	28	< LOQ
160	M4-PW	0	< LOQ
	M11-PW	14	< LOQ
	M18-PW	28	< LOQ
320	M5-PW	0	< LOQ
	M12-PW	14	< LOQ
	M19-PW	28	< LOQ
641	M6-PW	0	< LOQ
	M13-PW	14	0.223
	M20-PW	28	0.424
1280	M7-PW	0	0.256
	M14-PW	14	0.462
	M21-PW	28	0.813

1 The limit of quantitation (LOQ) was 0.200 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.100 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2 Results were generated using Analyst versions 1.4.1 and 1.6.2. Manual calculations may differ slightly.

Table 7: Biological Results: Day 28 Adult Survival and Growth of Hyalella Azteca During a Life Cycle Toxicity Test Using Spiked Sediment

Mean Measured Concentration in Sediment (μg a.i./kg)	Percent Survival 1,5 (mean ± std)	Average Body Length (mm)2,3,4 (mean ± std)	Average Individual Dry Weight (mg)2,4 (mean ± std)
NegativeControl	96 ± 5.2	5.52 ± 0.10	0.90 ± 0.04
Solvent Control	99 ± 2.9	5.49 ± 0.28	0.91 ± 0.13
Pooled Control	98 ± 4.4	5.50 ± 0.19	0.90 ± 0.09
58	97 ± 4.9	5.57 ± 0.27	0.90 ± 0.15
109	98 ± 3.9	5.65 ± 0.17	0.94 ± 0.08
239	98 ± 5.8	5.64 ± 0.18	0.92 ± 0.10
475	97 ± 6.5	5.62 ± 0.07	0.90 ± 0.01
992	98 ± 3.9	5.65 ± 0.21	0.93 ± 0.06

1 Average survival in twelve replicates on Day 28.

2 Average length and weight of surviving adult amphipods in four replicates terminated on Day 28.

3 Body length was measured from the base of the first antenna to the tip of the third uropod along the curve of the dorsum.

4 There were no statistically significant differences (α> 0.05) using a Dunnett’s, one-tailed test (length and dry weight), between the treatment groups and the pooled control.

5 There were no statistically significant difference (α< 0.05) from the pooled control group, using a non-parametric Kruskal-Wallis test.

Table 8: Biological Results: Day 42 Adult Survival, Growth and Reproduction of Hyalella Azteca During a Life Cycle Toxicity Test Using Spiked Sediment

Mean Measured Concentration in Sediment (μg a.i./kg)	Percent Survival1 (mean ± std)	Average Body Length (mm)1,2 (mean ± std)	Average Individual Dry Weight (mg)1 (mean ± std)	Average Number of Young Per Surviving Female3 (mean ± std)
Negative Control	94 ± 9.2	6.20 ± 0.15	1.40 ± 0.13	26.6 ± 3.7
Solvent Control	99 ± 3.5	5.96 ± 0.16	1.28 ± 0.08	23.9 ± 4.8
Pooled Control	96 ± 7.2	--4	--4	25.2 ± 4.3
58	95 ± 7.6	5.95 ± 0.11*	1.22 ±0.08*	20.4 ± 4.2**
109	98 ± 7.1	6.19 ± 0.16	1.42 ±0.08	24.0 ± 3.5
239	95 ± 7.6	6.15 ± 0.08	1.41 ± 0.07	21.9 ± 3.6
475	95 ± 11	6.19 ± 0.09	1.41 ± 0.05	21.4 ± 5.9
992	95 ± 5.4	6.15 ± 0.13	1.33 ± 0.06	21.6 ± 5.4

1 Average survival, length and weight of adult amphipods in eight replicates terminated on Day 42.

2 Body length was measured from the base of the first antenna to the tip of the third uropod along the curve of the dorsum.

3 The number of young present in the test compartments was enumerated on Days 28, 35 and 42. The total number of young counted for each replicate was divided by the number of females surviving at the end of the test (Day 42).

4 Controls were statistically different. Treatment groups were compared to the negative control group.

* There was a statistically significant difference (p < 0.05) from the negative control group, using a Dunnett’s, onetailed test. The mean measured length and dry weight value was within the range of historical control data (range: 5.27-7.09 mm and 0.69-1.40 mg, respectively). Therefore, the differences were neither considered dose responsive nor biologically meaningful.

** There was a statistically significant difference (p < 0.05) from the pooled control group, using a Dunnett’s, onetailed test. The number of neonates produced per surviving female was within the range of historical control data (range: 7.7-29.1 neonates/surviving female). Therefore, the difference was neither considered dose responsive nor biologically meaningful.

Validity criteria fulfilled:

yes

Reference 7

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 Nov 2015 - 21 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ASTM E1706 (Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. OCSPP Guideline 850.SUPP 850.1760: Whole Sediment Life Cycle Toxicity Test with Chironomus spp.

Version / remarks:

September 2009

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: Control, 0.250, 0.500, 1.00, 2.00 and 4.00 μg a.i./mL stock solutions, samples were taken from each test concentration and control
- Sampling interval: on Days 0, 16 and 57
- Sample storage before analysis: no storage

PORE WATER
- Concentrations: Control, 0.250, 0.500, 1.00, 2.00 and 4.00 μg a.i./mL stock solutions, samples were taken from each test concentration and control
- Sampling interval: on Days 0, 16 and 57
- Sample storage before analysis: no storage

OVERLYING WATER
- Concentrations: Control, 0.250, 0.500, 1.00, 2.00 and 4.00 μg a.i./mL stock solutions, samples were taken from each test concentration and control
- Sampling interval: on Days 0, 16 and 57
- Sample storage before analysis: no storage

Vehicle:

yes

Remarks:

Acetone

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: The dosed sand was placed in a fume hood and the acetone was allowed to evaporate for approximately one hour and one hour and 30 minutes under the fume hood.. Than 30 g sand premixes were added to 931 grams of untreated formulated sediment in plastic Nalgene® bottles and mixed on a rotary mixer for approximately three and a half hours. Next, 967.7 g of untreated formulated sediment were added to the premixes to achieve a final weight of 1034.5 g.
- Method of mixing: mixed on a rotary mixer for approximately 40 h.
- Details of spiking: A volume of 6 mL of the appropriate stock solution was added to 30.0 g of sand in a glass beaker and stirred with a glass stir rod.
- Controls: yes, control and solvent control
- Equilibration time: 2 d prior to the introduction of the test organisms
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 6 mL of acetone to 30.0 g of sand
- Evaporation of vehicle before use: yes, for one hour and 30 minutes under the fume hood

Test organisms (species):

Chironomus dilutus (previous name: Chironomus tentans)

Details on test organisms:

TEST ORGANISM
- Common name: non-biting midge
- Source: Columbia Environmental Research Center, U.S. Geological Survey, Columbia, Missouri, USA
- Handling of egg masses and larvae: 12 midges larvae were transfered into test compartment; transfers were made below the water surface using wide-bore pipettes.
- Age of animals at beginning of exposure: Midges used in the test were first instar larvae, and were approximately 4-days old at test initiation; On Day 0 of the test, first instar midge larvae (up to 4 days post hatch) were added to test compartments until each test compartment contained 12 larvae. On Day 6 of the test, first instar midge larvae (up to 4 days post hatch) were added to auxiliary male test compartments until each test compartment contained a total of 12 larvae.
- Feeding during test: yes
- Food type: diet of Chlorella and a suspension of TetraMin® flake food daily after hatching of larvae; Midges were fed a suspension of TetraMin® flake food during the test.
- Amount: 1.5 - 3.0 mL of a 4 g/L Tetramin® suspension of flake food in water
- Frequency: daily, but not on the last days of the test

ACCLIMATION
- Acclimation period: 2 d

Study type:

laboratory study

Test type:

flow-through

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

57 d

Exposure phase:

larvae from first generation (P)

Hardness:

Overlying water:
141 ± 2.31 mg/L as CaCO3 (control)
137 ± 2.31 mg/L as CaCO3 (Solvent control)
179 - 183 ± 1.15 - 6.93 mg/L as CaCO3 (test item concetrations)

Test temperature:

Overlying water:
22.5 ± 0.18 °C (control)
22.5 ± 0.18 °C (solvent control)
22.5 - 22.6 ± 0.17-0.19 °C (test item concentrations)

pH:

Overlying water:
8.0 ± 0.18 (control)
8.0 ± 0.15 (solvent control)
8.0-8.1 ± 0.14-0.15 (test concentrations)

Dissolved oxygen:

7.5 ± 0.82 mg/L (control)
7.3 ± 0.94 mg/L (solvent control)
6.9 - 7.2 mg/L 0.94 ± 1.02 (test concentrations)

Ammonia:

0.358 ± 0.237 mg/L as NH3 (control)
0.214 ± 0.151 mg/L as NH3 (solvent control)
0.171 - 0.199 ± 0.150 - 0.207 mg/L as NH3 (test concentration)

Conductivity:

337 ± 19 µS/cm (control)
342 ± 21 µS/cm (solvent control)
343 - 346 ± 20-22 µS/cm (test concentration)

Nominal and measured concentrations:

Control, solvent control, 2.5, 5.0, 10, 20 and 40 μg a.i./kg (nominal)
Control, solvent control, 1.2, 2.8, 5.6, 12 and 33 μg a.i./kg (mean measured sediment concentration)
Control, solvent control, 0.039, 0.085, 0.151, 0.347 and 0.718 μg a.i./L (mean measured pore water concentration)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 300 mL glass beakers with stainless steel mesh screen covered holes on opposite sides of the compartment
- Sediment volume: 100 mL
- Overlying water volume: 175 mL overlaying water
- Depth of sediment and overlying water: sediment depth 3.5 cm in a representative test compartment and depth of overlying water 5.6 cm in a representative test compartment
- Aeration: no

EXPOSURE REGIME
- No. of organisms per container (treatment): 12 midge larvae
- No. of replicates per treatment group: 12 (additional 4 replicates for use as auxiliary male replicates)
- No. of replicates per control / vehicle control: 12 (8 for the evaluation of survival and growth + 2 for analytical determination)
- Feeding regime: daily
- Type and preparation of food: Tetramin® suspension of flake food in water (test)
- Amount of food: 1.5 -3.0 mL of a 4 g/L Tetramin® suspension of flake food in water

RENEWAL OF OVERLYING WATER
- Details on volume additions: each replicate beaker received at least two volume additions of dilution water per day
- Flow-rate: Each replicate beaker received at least two volume additions of dilution water per day. The dilution water was delivered into the stainless steel splitting chambers. Each stainless steel splitting chamber, containing 10 plastic syringes, delivers to one stainless steel diluter tank. Each syringe delivers to a single test compartment within the diluter tank. This type of flow passively forces the dilution water out through the holes in the sides of the beakers and exchanges the water overlying the sediment. Dilution water delivery volume was verified prior to test initiation. A check was made daily to inspect the proper operation of the diluter, with the exception of two days during the test, when the check was inadvertently not documented. Test compartments were labeled with the project number, test concentration and replicate.

OVERLYING WATER CHARACTERISTCS
- Type of water: freshwater; moderately-hard water
- Source of water: from 40 meters deep located on the Wildlife International site
- Filtration: water was passed through a sand filter and pumped into a 37800 L storage tank where the water was aerated with spray nozzles. Prior to use in the test system, the water was filtered to 0.45 μm and passed through a UV sterilizer.
- Alkalinity: 180 to 184 mg/L as CaCO3 (control); 176 to 184 mg/L as CaCO3 (solvent control); 176 to 190 mg/L as CaCO3 (test concentrations)
- Total organic carbon: < 1%
- Pesticides and Organics (µg/L): < 0.010: Aldrin; Gamma BHC – Lindane ; Alpha BHC ; Alpha Chlordane ; Beta BHC ; Endosulfan I ; HCB Heptachlor ; Heptachlor Epoxide ; Delta BHC ; Telodrin ; < 6.3: Bolstar Coumaphos; Malathion; Demeton-O; Guthion (Azinphos-methyl); Merphos; Demeton-S; Diazinon; Methyl Parathion; Dichlorvos; Dursban (Chlorpyrifos); Disulfoton; Naled; Mevinphos; Phorate ; EPN; Ronnel; Ethion; Ethoprop; Stirophos; Ethyl Parathion; Tokuthion; Famphur; Fensulfothion; Trichloronate; Fenthion; Trithion; < 0.021 Gamma Chlordane Dieldrin; p,p-DDE; Endrin; p,p-DDT; Endrin Ketone; Endosulfan Sulfate; p,p-DDD; o,p-DDT; o,p-DDE; o,p-DDD; Mirex < 0.052; Endosulfan II < 0.031; Endrin Aldehyde < 0.10; Methoxychlor < 0.10; Kepone < 0.21; Toxaphene < 1.0; Chlordane < 0.52
- Metals: < 0.200 Aluminum; Iron; < 0.0200: Antimony; Arsenic; Zinc; Selenium; < 0.0050: Manganese; Barium; Beryllium; Vanadium; Cadmium; Silver; Cobalt; < 0.0100: Nickel;Copper; < 0.50: Nitrate Nitrogen; Nitrite Nitrogen; Fluoride; Bromide < 2.5; Potassium 7.00; Calcium 35.8; Chloride 4.0; Chromium < 0.0150; Sodium 19.1; Sulfate < 5.0; Thallium < 0.0300; Lead < 0.0150; Mercury < 0.00020; Magnesium 13.8

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Natural freshwater sediment collected at West Bearskin Lake, Minnesota

HANDLING OF NATURAL SEDIMENT
- Storage conditions: sediment was stored refrigerated or frozen after receipt in the laboratory
- Treatment before use: sub-sample of sediment was dried to determine the percent moisture content, which was the used to adjust the amount of sediment that was dosed for the test.

CHARACTERIZATION OF SEDIMENT
- Particle size distribution
- % sand: 45
- % silt: 52
- % clay: 3
- Composition (if artificial substrate): Silt loam with Peat Moss
- Moisture: 51.0% at 1/3 bar
Further constituents
- Pesticides and Organics (μg/kg): < 2.2 Aldrin, Gamma BHC – Lindane, Alpha BHC, Gamma Chlordane, Alpha Chlordane, Endosulfan I, Heptachlor, Heptachlor Epoxide; < 4.5 Dieldrin Mirex, o,p-DDD ,o,p-DDE ,Endosulfan II , o,p-DDT ,Endosulfan Sulfate , p,p-DDD ,Endrin , p,p-DDE, Endrin Aldehyde , p,p-DDT; < 180 Guthion (Azinphos-methyl) , Coumaphos , Bolstar , Malathion Demeton-O , Merphos , Demeton-S , Diazinon , Methyl Parathion, Dichlorvos , Mevinphos , Disulfoton , Naled , Dursban (Chlorpyrifos) , Phorate , EPN , Ronnel , Ethion , tirophos, Ethoprop , Famphur , Ethyl Parathion , Tokuthion , Trichloronate , Fenthion , Trithion; < 2.4 Delta BHC, < 2.7 Beta BHC, < 6.1 HCB, < 18 Methoxychlor, < 45 Chlordane, < 19 Kepone, < 4.8 Endrin Ketone, < .2 Telodrin, < 88 Toxaphene, < 530 Fensulfothion
- Metals (mg/kg):(mg/L) Aluminum 13,300, Magnesium 5,420, Antimony < 5.20, Manganese 473, Arsenic < 6.35, Mercury < 50.0, Barium 71.3, Nickel 41.6, Beryllium < 1.30, Nitrate by IC (solid) < 4.0, Bromide by IC (solid) < 13.2, Nitrite by IC (solid) < 2.6, Cadmium < 1.30, Potassium 1,620, Calcium 4,720, Selenium 7.62, Chloride by IC (solid) < 26.5, Silver < 1.30, Chromium 34.0, Sodium 478, Cobalt 12.4 , Sulfate by IC (solid) 632, Copper 59.3 , Thallium < 7.80, Fluoride by IC (solid) < 2.6 , Vanadium 58.1, Iron 49,500 , Zinc 114, Lead 6.58
- Organic matter: 14.6%
- pH dry matter and/or whole sediment: 5.3 (in soil:water ratio 1:1)
- Total organic carbon (%): 8.5%
- Bulk density: 0.71 gm/cc
- CEC: 12.5 meq/100 g

OTHER TEST CONDITIONS
- Light quality: Fluorescent light tubes (emit wavelengths similar to natural sunlight)
- Photoperiod: Photoperiod of 16 h of light and 8 h of darkness with a 30 min transition period of low light intensity
- Light intensity: 743 lux at the surface of the water

EFFECT PARAMETERS MEASURED:
The test compartments were observed daily to make visual assessments of abnormal behavior (e.g. leaving the sediment, climbing the walls of the test compartment, swimming in the water column). On Day 16 four replicates of the test, 20-day old survived larvae were removed from the sediment using a 425 μm sieve, and the numbers of live or dead organisms were enumerated. The total number of adults emerged at the end of the test period was recorded. Primary egg masses were collected and the number of eggs per egg mass was determined. The egg masses were held for six days to determine hatchability. Mortality of the first generation organisms observed in the treatment groups over the course of the test. The remaining eight replicate test chambers in each treatment and control group will be retained for use in evaluating emergence and reproduction.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: no range-finding tets was performed, but results were based on the results of an existing 10-Day spiked sediment test

Reference substance (positive control):

no

Duration:

57 d

Dose descriptor:

LC50

Effect conc.:

> 33 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: based on concentrations in the sediment

Duration:

57 d

Dose descriptor:

NOEC

Effect conc.:

>= 33 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks:

Development time, development rate

Remarks on result:

other: based on the concentrations in the sediment

Duration:

57 d

Dose descriptor:

LOEC

Effect conc.:

> 33 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks:

Development time, development rate

Remarks on result:

other: based on the concentrations in the sediment

Details on results:

- Reproduction of test animals at end of exposure period (%): There were a total of 44, 43, 25, 38, 26, 35 and 28 mated females in the negative control, solvent control, 1.2, 2.8, 5.6, 12 and 33 μg a.i./kg mean measured sediment treatment groups
- Total primary egg masses: The average number of eggs per primary egg mass was 1593, 1523, 1588, 1353, 1597, 1735 and 1751 in negative control, solvent control, 1.2, 2.8, 5.6, 12 and 33 μg a.i./kg mean measured sediment treatment groups, respectively.
- Other: Detailed analytical and biological results are summarized within the tables 1-7 in section "Any other information on results incl. tables"

Reported statistics and error estimates:

The results of the test were based on mean measured concentrations in the sediment. Statistical analyses were performed to evaluate differences between treatment and control groups for each of the following endpoints: survival, growth (ash-free dry weight), emergence and reproduction. The Day 16 percent survival data precluded the calculation of an LC50 as all concentrations tested had greater than 50% survival. The no-observed-effect-concentration (NOEC) and lowest-observed-effect-concentration (LOEC) were determined by visual interpretation of the dose-response pattern and statistical analyses of the survival, growth, emergence, and reproduction data. All statistical analyses were performed using TOXSTAT version 3.5 or SAS version 8.2. The data from the negative and solvent control groups were compared using a t-test. There were no statistically significant differences between the negative and solvent control groups for survival or growth on Day 16, therefore the treatment groups were compared to the pooled control replicates. Day 16 survival and ash-free dry weight data was evaluated for normality (Shapiro-Wilk’s) and homogeneity of variances (Levene’s), α = 0.01. The Day 16 ash-free dry weight data was found to be normal and homogeneous; therefore, a Bonferroni test was used to identify statistically significant differences between the treatment groups and the pooled control. The assumption of normality was not met in the survival data, so an attempt was made to correct the condition by an arc sine square root transformation of the data. Since the transformation did not correct the problem, the data in the treatment groups were compared to the pooled control data using a Kruskal-Wallis non-parametric test to identify any significant differences (α = 0.05). The emergence ratio and development rate were calculated for each replicate of each control and treatment group using the TOXSTAT version 3.5 and SAS System for Windows Version 9.4.

Analytical Results:

Table 1: Measured Concentrations of the test item in Stock Solution Samples

Nominal Concentration (μg a.i./mL)	Sample Number (149A-253A-)	Measured Concentration (μg a.i./mL)	Percent of Nominal (1)
0.25	ST-1	0.225	90.0
0.50	ST-2	0.451	90.1
1.0	ST-3	0.966	96.6
2.0	ST-4	1.77	88.7
4.0	ST-5	3.39	84.9

(1) Results were generated using OpenLab version A.01.05. Manual calculations may differ slightly.

 

Table 2: Measured Concentrations of the test item in Sediment Samples from a Life-Cycle Toxicity Test with Chironomus dilutus

Nominal Test Concentration (μg a.i./kg)	Rep.	Sample Number (149A-253A-)	Sampling Time (Days)	Measured Concentration (μg a.i./kg)1,2,3	Percent of Nominal2	Mean Measured Concentration4 (μg a.i./kg)	Mean Measured Percent of Nominal
Negative	M	1-SED	0	<LOQ	--	--	--
Control	N	8-SED	16	<LOQ	--
	O	15-SED	57	<LOQ
Solvent	M	2-SED	0	<LOQ	--	--	--
Control	N	9-SED	16	<LOQ		--
	O	16-SED	57	<LOQ	--
2.5	M	3-SED	0	1.61	64.6	1.2±0.614	48
	N	10-SED	16	1.42	56.9	CV=52.7%
	O	17-SED	57	<LOQ	--
5.0	M	4-SED	0	4.47	89.4	2.8±1.59	56
	N	11-SED	16	2.76	55.1	CV=55.8%
	O	18-SED	57	1.30	26.1
10	M	5-SED	0	7.48	74.8	5.6±2.77	56
	N	12-SED*	16	6.81	68.1	CV=49.9%
	O	19-SED	57	2.38	23.8
20	M	6-SED	0	16.6	82.8	12±3.94	60
	N	13-SED**	16	11.7	58.6	CV=31.8%
	O	20-SED	57	8.81	44.0
40	M	7-SED	0	34.2	85.4	33±0.971	83
	N	14-SED	16	32.3	80.8	CV=2.91%
	O	21-SED	57	33.6	84.0

1 The limit of quantitation (LOQ) was 0.928μg a.i./kg, calculated as the product of the concentration of the lowest calibration standard (0.0200μg a.i./L) and the dilution factor of the matrix blank samples (20.0), divided by the soil content of the negative control from day 0 (0.431).

2 Results were generated using Analyst version 1.4.1 and Excel 2010 in full precision mode. Manual calculations may differ slightly.

3 Analytical results were generated using wet weights. The tabulated values are reported on a dry weight basis.

4 Where measured value was < LOQ, the value of ½ the LOQ was used for the mean measured concentration calculation.

* Sample 149A-253A-12-SED recovery was not within the range of the calibration curve. The sample was re-diluted and reanalyzed in duplicate. The mean result of the reanalysis is reported above.

**Sample 149A-253A-13-SED recovery was not within the range of the calibration curve The sample was re-diluted and reanalyzed in duplicate. The reanalysis results did not match so the sample was re-extracted. The result of the reextraction is reported above.

 

The measured concentrations of the main metabolite in Sediment Samples from a Life-Cycle Toxicity Test with Chironomus dilutus was below the LOQ (2.32 μg a.i./kg) except for the highest test item (40 µg a.i./kg) concentration. Here a measured concentration of 3.72 and 5.32 µg a.i./kg was determined.

 

The measured Concentrations of the test item in Overlying Water Samples from a Life-Cycle Toxicity Test with Chironomus dilutus was below the LOQ (0.0400 μg a.i./L) for all test item concetrations. This was also the case for the measured concentration of the metabolite in the overlaying water. The only measurable concentrations were at 2.5 µg a.i/kg with 0.101 µg a.i./L and at 10 µg a.i./kg with 0.107 µg a.i./L.

  

Table 3: Measured Concentrations of test item in Pore Water Samples from a Life-Cycle Toxicity Test with Chironomus dilutus

Nominal Test Concentration (μg a.i./kg)	Rep.	Sample Number (149A-253A-)	Sampling Time (Days)	Measured Concentration (μg a.i./L)1,2	Mean Measured Concentrations (μg a.i./L)
Negative	M	1-PW	0	<LOQ	--
Control	N	8-PW	16	<LOQ
	O	15-PW	57	<LOQ
Solvent	M	2-PW	0	<LOQ	--
Control	N	9-PW	16	<LOQ
	O	16-PW	57	<LOQ
2.5	M	3-PW	0	0.05233	0.039
	N	10-PW	16	0.0451
	O	17-PW	57	<LOQ
5.0	M	4-PW	0	0.1043	0.085
	N	11-PW	16	0.0963
	O	18-PW	57	0.0545
10	M	5-PW	0	0.2063	0.151
	N	12-PW	16	0.172
	O	19-PW	57	0.0763
20	M	6-PW	0	0.4883	0.347
	N	13-PW*	16	0.428
	O	20-PW	57	0.125
40	M	7-PW	0	0.8373	0.718
	N	14-PW**	16	0.805
	O	21-PW	57	0.512

1) The limit of quantitation (LOQ) was 0.0400 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0200 μg a.i./L) and the dilution factor of the matrix blank samples (2.00). Where measured value was <LOQ, the value of ½ the LOQ was used for the mean measured concentration.

2) Results were generated using Analyst version 1.4.1. Manual calculations may differ slightly.

3) Samples 3-PW through 7-PW were initially diluted below the calibration curve. Samples were re-diluted and reanalyzed. The results of the reanalysis are reported above.

* Sample was reanalyzed. The original analysis result (0.431 μg a.i./L) was not within the calibration curve.

**Sample was reanalyzed. The original analysis result (0.902 μg a.i./L) was not within the calibration curve

The measured concentration for the metabolite in Pore Water Samples were below the LOQ (0.100 μg a.i./L) except at a test item concentration of 5.0 µg a.i./kg with a measured concentration of 0.112 µg a.i./L.

Biology Results:

Table 4: Day 16 Adult Survival and Growth of Chironomus dilutes During a Life Cycle Toxicity Test Using Spiked Sediment

Mean Measured Concentration in Sediment (μg a.i./kg)	Percent Survival 1,2 (mean ± std)	Average Individual Ash-Free Dry Weight (mg) 1,2 (mean ± std)
Negative Control	100±0.0	2.45 ± 0.15
Solvent Control	96± 4.6	2.59±0.06
Pooled Control	98±3.7	2.52±0.13
1.2	87±8.5	2.67±0.24
2.8	100±0.0	2.58±0.16
5.6	100±0.0	2.23±0.16
12	100±0.0	2.47±0.19
33	96±8.5	2.47±0.42

1 average percent survival and ash-free dry weight of surviving organisms in four replicates terminated on Day 16.

2 Calculated using TOXSTAT version 3.5, manual calculations may differ slightly. There were no statistically significant differences (α = 0.05) for survival or weight from the pooled control group using a Kruskal-Wallis test or Bonferroni t-test.

  

Table 5: Reproduction of Chironomus dilutes During a Life Cycle Toxicity Test Using Spiked Sediment

Mean Measured Concentration in Sediment (μg a.i./kg)	Total Number of Mated Females per Treatment Group1	Total Number of Primary Egg Masses Produced per Treatment Group 1	Number of Primary Egg Masses per Mated Female 2	Mean Time to Oviposition (Days) 2	Average Number of Eggs per 1º Egg Mass 2	Average Number of Eggs per Mated Female 2	Average Percent Hatch of 1º Egg Masses Produced per Treatment Group 2
Negative Control	44	24	0.535 (± 0.204)	2.06 (± 2.04)	1593 (± 481)	864 (± 312)	86.7 (± 18.2)
Solvent Control	43	34	0.811 (± 0.205)	1.52 (± 0.45)	1523 (± 359)	1260 (± 460)	77.2 (± 17.5)
Pooled Control	87	58	--3	1.79 (± 1.46)	1556 (± 406)	1075 (± 435)	81.6 (± 17.9)
1.2	25	16	0.646 (± 0.226)	1.78 (± 0.90)	1588 (± 551)	1002 (± 429)	92.9 (± 12.9)
2.8	38	26	0.669 (± 0.197)	2.21 (± 2.36)	1353 (± 295)	973 (± 304)	81.6 (± 11.3)
5.6	26	22	0.844 (± 0.186)	1.41 (± 0.43)	1597 (± 568)	1350 (± 646)	91.0 (± 10.1)
12	35	22	0.665 (± 0.260)	1.31 (± 0.46)	1735 (± 455)	1192 (± 613)	79.6 (± 22.8)
33	28	25	0.925 (± 0.149)	1.39 (± 0.40)	1751 (± 473)	1658 (± 589)	84.9 (± 12.7)

There were no statistically significant differences (α= 0.05) between the negative or pooled control group and any of the treatment groups, using a Bonferroni or Dunnett’s test.

1Calculated using Excel 2010, in full-precision mode. Manual calculations may differ slightly. No statistical analysis was performed.

2Calculated using SAS version 9.4 and TOXSTAT version 3.5. Manual calculations may differ slightly.

3Controls were statistically different and should not be pooled. Treatment groups were compared to the Negative Control group

 

 

Table 6: Summary of Emergence and Development of Midges (Chironomus dilutus) During the 57-Day Exposure to Sediment-Incorporated test item

Mean Measured Concentration in Sediment	Number Emerged 1			Mean Emergence Ratio 2,4	Mean Development Time 4	Mean Development Rate 3,4	Mean Time to Males Females Total Death 4
(μg a.i./kg)	Males	Females	Total		(Days)		(Days)
Negative Control	44	44	88	0.917 (± 0.100)	25.9 (± 2.30)	0.0406 (± 0.004)	3.41 (± 0.50)
Solvent Control	31	49	80	0.833 (± 0.109)	25.4 (± 1.40)	0.0415 (± 0.002)	4.13 (± 0.70)
Pooled Control	75	93	168	0.875 (± 0.110)	25.6 (± 1.85)	0.0411 (± 0.003)	--5
1.2	33	33	66	0.688 (± 0.139)*	28.9 (± 4.76)	0.0373 (± 0.006)	3.57 (± 0.97)
2.8	35	41	76	0.792 (± 0.167)	26.3 (± 2.57)	0.0406 (± 0.004)	3.60 (± 0.71)
5.6	46	33	79	0.823 (± 0.137)	25.7 (± 2.99)	0.0410 (± 0.005)	3.39 (± 0.55)
12	39	39	78	0.813 (± 0.146)	25.5 (± 1.71)	0.0417 (± 0.002)	3.32 (± 0.52)
33	50	29	79	0.823 (± 0.175)	24.4 (± 1.84)	0.0429 (± 0.003)	3.73 (± 0.68)

1 Each replicate contained 12 midge larvae at test initiation, for a total of 96 larvae per control and treatment group.

2 Emergence ratio is calculated as the number of emerged midges divided by the initial number exposed, and corresponds to percent emergence.

3 The development rate represents that portion of larval development which takes place per day.

4 Calculated using SAS version 9.4 or TOXSTAT version 3.5. Manual calculations may differ slightly.

5 Controls were statistically different and should not be pooled. Treatment groups were compared to the Negative Control group.

* Indicates a statistically significant difference in comparison to the pooled control (α= 0.05) using a Bonferroni t-test; however, not considered to be biologically meaningful. There were no statistically significant differences for development time, development rate or mean time to death (α= 0.05) between the negative or pooled control group and any of the treatment groups, using a Bonferroni or Dunnett’s test.

Validity criteria fulfilled:

yes

Description of key information

Freshwater:

EC10 (28 d) = 5.96 µg a.s./kg dw sediment (key, M-509624-01-1, Chironomus riparius, OECD 218, emergence rate, initial measured concentration)

NOEC (28 d) = 6.76 µg a.s./kg dw sediment (key, M-509624-01-1, Chironomus riparius, OECD 218, emergence rate, initial measured concentration)

EC10 (28 d) = 0.71 µg a.s./L (key, M-532889-01-1, Chironomus riparius, OECD 219, emergence rate, nominal)

NOEC (28 d) = 0.8 µg a.s./L (key, M-532889-01-1, Chironomus riparius, OECD 219, emergence rate, nominal)

EC50 (48 h) = 230 µg a.s./L (key, M-518365-01-1, Chironomus riparius, OECD 235, mobility, nominal)

Marine water:

NOEC (10 d) ≥ 728 µg a.s./kg dw sediment (key, M-542852-01-1, Leptocheirus plumulosus, OPPTS 850.1740, mean measured concentration)

LC50 (10 d) > 728 µg a.s./kg dw sediment (key, M-542852-01-1, Leptocheirus plumulosus, OPPTS 850.1740, mean measured concentration)

Key value for chemical safety assessment

EC50 or LC50 for marine water sediment:

728 µg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

5.96 µg/kg sediment dw

Additional information

Several experimental guideline studies are available investigating the toxicity of the test item towards freshwater and marine sediment organisms according to GLP. Two long-term studies were performed with the freshwater species Chironomus riparius (M-509624-01-1 and M-532889-01-1), two long-term studies were performed with Chironomus dilutus (M-542839-02-1 and M-560359-02-1) and one with the marine species Leptocheirus plumulosus (M-542852-01-1), respectively. One short-term study was conducted with the freshwater species Chironomus riparius (M-518365-02-1). Due to a low solubility of the test item, acetone or dimethylformamide (DMF) were used as solvents for application of test item to either sediment or water compartment.

In the key study Chironomus riparius (M-509624-01-1) was exposed to initial nominal concentrations of 2.50, 5.00, 10.0, 20.0, 40.0 and 80.0μg a.s./kg dw in a static test system for 28 d according to OECD 218. Spiked sediments of the test concentrations were prepared by addition of stock solutions of the test substance directly into the sediment. Stock solutions of the test substance dissolved in acetone were used. Suitable amounts of these stock solutions were mixed with suitable amounts of fine quartz sand for each test concentration and were afterwards mixed with the suitable amounts of sediment per test concentration. During the study, the measured concentrations of the test item and its metabolite were analysed four times (on day 0, 4, 8 and 28) in the sediment, the overlying water and pore water, in all test levels and the control(s). Additional measurements of sediment samples were made on day -2 for all test concentrations and the controls directly after sediment spiking. Chemical analyses of the sediment, overlying water and pore water show only a small partitioning of the test item from the sediment into the water phase over time (max. 12.5% were found in the overlying water and max. 1.9% in the pore water). Initially measured test concentrations (day 0) in the sediment (in µg a.s./kg dw sed) were used for reporting and evaluation of the results. Analyses of the sediment over time showed recoveries of the test item ranging from 63.7% to 79.2% (mean = 69.6%) of nominal for all test concentrations on day 0. On day 4, 59.6% to 76.4% (mean = 65.7%), on day 8, 53.3% to 67.6% (mean = 58.8%) and on day 28, 42.7% to 54.4% (mean = 47.4%) of nominal were found in the sediment, respectively. Analyses of the overlying water showed recoveries of the test item of 9.6 % to 11.1% (mean = 10.6%) of nominal applied amount of a.s. per test concentration on day 0. On day 4, 10.9% to 12.5% (mean = 11.5%), on day 8, 9.9% to 11.1% (mean = 10.4%) and on day 28, 0% to 5.4% (mean = 3.3%) of nominal were found, respectively. Emergence, sex and development rates were determined daily. A statistically significant difference in emergence was determined for initial measured test concentrations from 12.73 to 59.75 µg a.s./kg dw sediment as compared to the pooled controls (solvent control + test medium control), resulting in a NOEC (28 d) of 6.76 µg a.s./kg dw sediment. The EC10 (28 d) for the emergence rate was determined to be 5.96 µg a.s./kg dw sediment. For the development rate (pooled sex) a statistically significant difference was found for initial measured test concentrations of 12.73 µg a.s./kg dw sediment as compared to the pooled controls, resulting in a NOEC (28 d) of 6.76 µg a.s./kg dw sediment. The EC10 (28 d) for the development rate was determined to be 10.7 µg a.s./kg dw sediment. All validity criteria were met and thus, the test is considered valid.

A second long-term study with Chironomus riparius was performed according to OECD 219 with spiked water application (M-532889-01-1). The test species was exposed to initial nominal concentrations in the overlying medium of 0.20, 0.40, 0.80, 1.60, 3.20 and 6.40μg a.s./L in a water-sediment test system for a period of 28 d. Test item was dissolved in dimethylformamide (DMF) and appropriate amounts were applied in the overlying water column. During the study, the measured concentrations of the test item and its metabolite were analysed in the overlying water of the sediment water four times, on day 0 (1 hour), day 4, day 8 and day 28 in all test levels and in the controls (test medium and solvent control). Analyses of the overlying water at the beginning of the exposure period (nearly one hour after spiking) reflect high recoveries of the test item with 108% to 114% (mean 110%) of nominal concentrations in all test levels. Thus, all results and are based on nominal concentrations of the test item in the overlying water. Exposure recoveries of the test item in the overlying water of all test concentrations were found to be 52-57% (mean 54%) after 4 d, 22-31% (mean 27%) after 8 d and 0-1.8% (mean 1.7%) after 28 d. Chemical analysis of the metabolite (averages) over time yield to 0.5% of nominal on day 0, 16% on day 4, 17% on day 8 and 4.4% on day 28. The chronic effect of the test item on development and emergence of C. riparius exposed over 28 d can be quantified by an overall-NOEC (28 d) of 0.80 µg a.s./L (based on nominal initial concentrations). The lowest EC10 (28 d) of 0.71 µg a.s./L was derived for emergence rate. All validity criteria were met and thus, the test is considered valid.

A third long-term study with Chironomus was performed testing the species Chironomus dilutus according to EPA 600/R-99/064 and OCSSP draft guideline 850.SUPP. with spiked sediment (m-560359-02-1). The test organisms were exposed to a geometric series of five test concentrations of 2.5, 5.0, 10 and 40 µg a.i./kg sediment, a solvent control and a negative control for 57 d under flow-through test conditions. The test item was dissolved in acetone and a dilution series was prepared in acetone. Appropriate volume of the stock solutions was applied in the sediment, mixed and allowed to evaporate. After mixing the batch sediments, test compartments were prepared by adding specific volume of the appropriate dosed sediment to the corresponding replicates. Each test compartment contained sediment and overlying water. The mean measured concentrations of the substance in the sediment, based on the average sediment concentration determined from analyses conducted on Days 0, 16 and 57 of the exposure were 1.2, 2.8, 5.6, 12 and 33μg a.i./kg. The results of the test are based on the mean measured concentrations of the substance in the sediment. On Day 16, there were no statistical differences observed on survival or growth relative to the pooled control group in any of the treatment groups. At test termination (Day 57) there were no biologically meaningful statistical differences on any of the emergence or reproductive endpoints when the treatment groups were compared to the negative or pooled control. Based on the results of this study, the LOEC was >33μg a.i./kg mean measured dry weight of sediment and the NOEC was 33μg a.i./kg mean measured dry weight of sediment. The 16 and 57-Day LC50 were >33μg a.i./kg mean measured dry weight of sediment the highest concentration tested.

Chironomus dilutus was selected to investigate the short-term toxicity of the test item to sediment organisms (M-542839-02-1). The study was performed according to EPA OPPTS 850.1735. The test organisms were exposed to a geometric series of six test concentrations of 10, 20, 40, 80, 160 and 320 µg test item/kg of dw of the sediment, a solvent control and a negative control for 10 d under flow-through test conditions. The test item was dissolved in acetone and a dilution series was prepared in acetone. Appropriate volume of the stock solutions was applied in the sediment, mixed and allowed to evaporate. Afterwards the treated sediment was mixed with untreated sediment to achieve a final weight of 1400 grams. After mixing the batch sediments, test compartments were prepared by adding approximately 100-mL of the appropriate dosed sediment to the corresponding replicates. Each test compartment contained sediment and overlying water. The measured concentrations of the test item in the 1.00, 2.00, 4.00, 8.00, 16.0 and 32.0 µg a.s./mL stock solutions yielded 91.2, 90.9, 89.0, 91.3, 91.1 and 88.3% of the nominal values, respectively. The results of the study are based on mean measured test concentrations in the sediment. Observations of mortality and abnormal behavior were made daily during the test. Survival and growth (AFDW = ash-free dw) were determined at the end of the 10-day test period. There were test substance related effects observed on survival. The LC50 (10 d) for survival, the most sensitive endpoint, was 34 µg a.s./kg dw sediment, based on mean measured concentrations in the sediment. The NOEC (10 d) for survival was 11 µg a.s./kg dw sediment, based on mean measured concentrations in the sediment. All validity criteria were met and thus, the test is considered valid.

The fifth long-term study was performed with Hyalella azteca according to the draft OCSPP guidance 850.1770, ASTM guidance of E1706 and the EPA guidance of 600/R-99/064 with spiked sediment (M-560357-02-1). The species was exposed to the nominal concentrations of 80, 160, 320, 641 and 1280μg a.i./kg of sediment based on the dry weight of the sediment for 28 days, then moved to clean water for 14 days. The mean measured concentrations of the test item in the sediment, based on the average sediment concentration determined from analyses conducted on Days 0, 14 and 28 of the exposure were 58, 109, 239, 475 and 992μg a.i./kg, respectively. The results of the test are based on the mean measured concentrations of the substance in the sediment. The 42-day LC50 value for Hyalella azteca exposed to the test item in sediment was >992μg a.i./kg (>50.0 μg a.i./L), the highest concentration tested, based on mortality data. There were no biologically meaningful statistically significant differences between the negative or pooled control and the treatment groups for the Day 28 survival, Day 42 survival, growth or reproduction. Therefore, the Day 42 LOEC for all endpoints was determined to be >992 μg a.i./kg (>50.0 μg a.i./L) and the Day 42 NOEC was 992μg a.i./kg (50.0 μg a.i./L).

In a short-term exposure according to OECD 235 Chironomus riparius was exposed to nominal concentrations of 8.00, 16.0, 32.0, 64.0, 128 and 256μg a.s./L in a static system for 48 h (M-518365-02-1). Three replicates, containing 10 animals each, were tested for each test item concentration and the controls (test medium and solvent control). Test item was dissolved in dimethylformamide (DMF) and appropriate amounts were applied in the overlying water column. The analysed active substance concentration found in all freshly prepared test levels on day 0 ranged from 104 to 123% (average 112%) compared to nominal. Analysed concentrations were comparable in aged test solutions on day 2 (95 - 115% (average 102%) of nominal). Due to the high recoveries at the beginning of the exposure and the analytical findings after 2 d, all results were based on nominal concentrations. Immobilisation of C. riparius was recorded resulting in an EC50 (48 h) of 230 µg a.s./L (nominal, 95% CL: 162 - 414 µg a.s./L). A NOEC (48 h) of 32 µg a.s./L (nominal) was calculated. All validity criteria were met and thus, the test is considered valid.

In the only study with marine species groups of the amphipods Leptocheirus plumulosus was exposed to a geometric series of five test concentrations, a solvent control and a negative control for 10 d under static test conditions (M-542852-01-1). Five replicates were maintained in each treatment and control group. The test item was dissolved in acetone and a following dilution series was also performed in acetone. An appropriate amount of these stock solutions were applied in the sediment to achieved the appropriate test item concentration in the sediment. Two additional replicates were added in each treatment and control group for analytical sampling of water and sediment. The analytical replicates sampled on day 0 contained no amphipods, while amphipods were added at test initiation to the analytical replicates sampled on day 10. Each test compartment contained sediment and overlying water. Nominal definitive test concentrations of 100, 200, 400, 800 and 1600 µg test item/kg dw sediment (corresponding to mean measured concentrations of 45, 103, 199, 356 and 728 µg a.s./kg dw sediment) were tested. The results of the study were based on mean measured test concentrations in the sediment. Observations of mortality and abnormal behavior were made daily during exposure. Survival was determined at the end of the 10d test period. ALC50 (10 d) of >728 µg a.s./kg dw sediment based on mean measured concentrations. The LOEC and NOEC were >728 and ≥ 728 µg a.s./kg dw sediment, respectively, based on mean measured concentrations. All validity criteria were met and thus, the test is considered valid.