N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Sep 1998 to 18 Dec 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: BBA Guideline Proposal

Version / remarks:

1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline, proposal for toxicity test with Chironomidae

Version / remarks:

May 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of an appropriate volume of the aqueous test solution, i.e. 30 mL samples from each beaker per concentration and the control were taken from the centre of the test vessels at day 0, 2, 7, 14, 28 and 0, 3, 7, 14, 28 for exposure scenarios A and B respectively. These samples were analysed and the concentration the test substance measured.
At the beginning, after 7 days and the end of the experiment, the concentration of the test substance in the sediment was measured in test vessels with the highest test concentrations and the lower one, i.e. at dose levels 0.0040, 0.0020 mg/L and 0.32, 0.16 mg/kg dry sediment for exposure scenarios A and B, respectively. To monitor changes in sediment or interstitial water chemistry during the course of the experiment, separate sediment chemistry chambers were set up and sampled at the start and after 7 days of the experiment.

Vehicle:

yes

Remarks:

culture medium (exposure scenario A); Acetone (exposure scenario B)

Details on sediment and application:

PREPARATION OF SPIKED WATER
- Preparation of stock solutions: 20.4 mg test substance were mixed and made up to 100 mL with reconstituted water to prepare stock solution 1. Stock solutions 2-5 were prepared by mixing appropriate stock solution 1 and making up to 100 mL with M4-Medium at test day 0 of exposure. The final test solutions were prepared by adding 10 mL of these stock solutions into the test chamber containing 560 mL M4- medium.
- Application: The stock solutions were added to the water column of the test vessels below the water surface by using a pipette and gently mixing the upper water layer to ensure homogeneous distribution without disturbing the sediment.
- Controls: 10 mL culture medium (M4 according to Elendt) was added at test initiation.

PREPARATION OF SPIKED SEDIMENT
- Preparation of stock solutions: 13.32 mg test substance were mixed and made up to 100 mL with acetone to prepare SL l. 5.0 mL of this stock solution were mixed and made up to 10 mL with acetone to prepare SL 2. 5.0 mL of stock solution 2 were mixed and made up to 10 mL with acetone to prepare SL 3. 5.0 mL of stock solution 3 were mixed and made up to 10 mL with acetone to prepare SL 4. 5.0 mL of stock solution 4 were mixed and made up to 10 mL with acetone to prepare SL 5. 5.0 mL of stock solution 5 were mixed and made up to 10 mL with acetone to prepare SL 6.
- Application: After evaporation of the acetone the dry sand was added to the aged sediment (1100 g sediment equivalent to 815 g dry sediment) to achieve the planned test concentrations and thoroughly mixed with a Mixer for about 30 minutes.
- Controls: Aged Sediment with acetone and without acetone treated sand were used as controls

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Culture condition: Chironomus larvae are reared in crystallizing dishes spread at the bottom with a thin layer of Kieselgur of about 1 - 2 cm depth and covered with M4 medium to a height of about 6 cm. The rearing vessels are kept in a suitable cage to prevent escape of emerging adults. Culturing is performed in a constant environment room at 20 ± 2°C, a photoperiod of 16 hours light and 8 hours dark (transition period 30 min.) and relative air humidity of 60 - 90%. Gentle aeration is provided throught the culture period.
- Feeding: The Chironomus larvae are fed with a fish flake food (dry ground powder of TETRAMIN). Some green algae (Scenedesmus subspicatus) are added when new culture vessels are set up and sometimes throughout culturing.
- Handling: Once adults are present within the breeding cage, all larvae rearing vessels are checked 3 times a week for deposition of the gelatinous egg masses. If present, the egg packages are removed carefully and will be used to start new rearing vessels or for toxicity tests.
- Age of animals at beginning of exposure: 1st instar larvae, 2-3 days old
- Handling of Chironomus larvae used in the test: The 1st instar larvae used in this study are obtained by transferring fresh egg masses to small crystallizing dishes with culture medium (M4-medium). After 2-3 days the L1 larvae have hatched and are transferred to the test beakers.
- Feeding of Chironomus larvae during the test: A commercially available fishfood (TETRAMIN) is used as food. 1 g Tetramin is ground finely and suspended in 20 mL water. From this stock a certain amount is added to the test vessel in order to provide each larvae with about 1 mg food per day during the course of the experiment.

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

Remarks:

Exposure scenario A and B

Hardness:

- Exposure scenario A: 232 - 260 mg/L as CaCO3
- Exposure scenario B: 244 - 248 mg/L as CaCO3

Test temperature:

- Exposure scenario A: 20.7 - 21.9 °C
- Exposure scenario B: 19.5 - 21.1 °C

pH:

- Exposure scenario A: 7.2 - 8.6
- Exposure scenario B: 7.7 - 8.6

Dissolved oxygen:

- Exposure scenario A: 3.9 - 10.7 mg O2/L
- Exposure scenario B: 3.6 - 9.0 mg O2/L

Conductivity:

- Exposure scenario A: 656 - 735 μS/cm
- Exposure scenario B: 685 - 712 μS/cm

Nominal and measured concentrations:

Nominal concentrations:
- Exposure scenario A: 0 (control), 0.000125, 0.00025. 0.00050. 0.0010, 0.0020 and 0.0040 mg/L
- Exposure scenario B: 0 (control), 0.010, 0.020. 0.040. 0.080. 0.16 and 0.32 mg/kg dry sediment
Measured concentrations:
- Exposure scenario A: - Exposure scenario B: 0 (control), 0.0003, 0.0003, 0.0004, 0.0003. 0.0003 and 0.0004 mg/L, respectively, at test day 0 (in water phase); 0.12 mg/kg sediment (wet) and 0.24 mg/kg sediment (wet) for the nominal 0.16 and 0.32 mg/kg dry sediment treatments, respectively.

Details on test conditions:

TEST SYSTEM (Exposure scenario A)
- Test container: 1 L glass beakers with a diameter of 9 cm
- Sediment weight: 120 g sediment (moist weight)/container
- Overlying water weight: 540 g/ container
- Depth of sediment and overlying water: M4 medium was used according to Guideline recommendations. The water column had a depth of about 8 cm (at the end of exposure 6 cm). The sediment layer was about 1 - 2 cm thick.
- Aeration: Yes
- Aeration frequency and intensity: Gentle aeration was provided through a glass Pasteur pipette situated about 2 - 3 cm above the sediment layer. After addition of the larvae aeration was stopped for the following 24 hours.
- Others: The water level was marked outside on the test beaker. The test beakers were then covered with parafilm with small holes to reduce evaporation throughout the test and to allow collection of emerged midges. The test beakers were prepared 8 days before the start of the denitive test (test substance application) to allow stabilization of the systems under test conditions.

TEST SYSTEM (Exposure scenario B)
- Test container: 1 L glass beakers with a diameter of 9 cm
- Sediment weight: 160 g sediment (moist weight)/container
- Overlying water weight: 500 g/ container
- Depth of sediment and overlying water: M4 medium was used according to Guideline recommendations. The water column had a depth of about 8 cm (at the end of exposure 6 cm). The sediment layer was about 1.6 cm thick.
- Aeration: Yes
- Aeration frequency and intensity: A gentle aeration was carried out for 1.5 hours before start of exposure. After addition of the larvae aeration was stopped for the following 24 hours. Following gentle aeration was provided through a glass Pasteur pipette situated about 2 - 3 cm above the sediment layer.
- Others: The water level was marked outside on the test beaker. The test beakers were then covered with parafilm with small holes to reduce evaporation throughout the test and to allow collection of emerged midges.The test beakers were prepared 48 hours before the start of the denitive test (test substance application) to allow stabilization of the systems under test conditions.

EXPOSURE REGIME (Exposure scenario A and B)
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 3
- No. of replicates per control / vehicle control: 3

OVERLYING WATER CHARACTERISTCS
- OECD 219 M4 medium

CHARACTERIZATION OF ARTIFICIAL SEDIMENT (Exposure scenario A and B)
Artificial sediment was prepared according to Guideline recommendations containing:
- % peat: 5.5
- % kaolin clay: 20
- % industrial sand: 74.5 (fine sand with more than 50% of the particles between 50 and 200 microns)
- pH: adjusted to 6.9 (exposure scenario A) and 6.7 (exposure scenario B) by adding chemically pure calcium carbonate
- OC: 1.7%

OTHER TEST CONDITIONS (Exposure scenario A and B)
- Photoperiod: 16:8 h (light : dark), 30 minutes transition period
- Light intensity: 800 - 1000 LUX

EFFECT PARAMETERS MEASURED
Visual assessments (behavior, mortalities, emergence) were made daily (except for test days 4, 5, 12, 19, 25 and 26 for exposure scenario A and 3, 4. 10, 11, 18, 24 and 25 for exposure scenario B). The number, time and sex of emerged adults was recorded. Afterwards adults were removed from the test vessels. The weight of larvae incubated for 10 days under exposure scenario A and incubated for 8 days under exposure scenario B were measured after sieving the sediment from each test beaker using a 250 µm sieve. The dry weight of the larvae was measured after drying at 60 ˚C to a constant weight.

RANGE FINDING STUDY
Prior to initiating the definitive tests, RANGE-FINDING tests were performed:
- Exposure scenario A: 0.01, 0.1, 1.0 mg/L and 0.0001, 0.001, 0.01 mg/L
- Exposure scenario B: 0.01. 0.1, I mg/kg dry sediment and 10, 100 mg/kg dry sediment
Preliminary test with test concentrations of 0.50, 1.0, 2.0, 4.0, 8.0 and 16 mg/kg dry sediment.
Based on the results of the range finding tests and the preliminary test, the nominal test concentrations were selected.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: spiking of the water column

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.04 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

emergence rate

Remarks on result:

other: spiking of the sediment

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.004 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks on result:

other: spiking of the water column

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.08 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

development rate

Remarks on result:

other: spiking of the sediment

Details on results:

An overview of the results is provided in Table 1 - Table 3 in 'Any other information on results incl. tables'.
- Analysis and calculation of the concentration of the test substance in test water and the sediment compartment (Exposure scenario A): The nominal test concentrations were 0.000125, 0.00025, 0.00050, 0.0010, 0.0020 and 0.0040 mg/L. The actual measured test concentrations in the water phase were < LOD, < LOD, < LOD, 0.00026, 0.00044 and 0.00049 mg/Lat test day 0 (1-3 hours after application). At the end of the test (test day 28) water concentrations were < LOD for all test concentrations. Analytical results of very short disappearance of the test substance in this test were in conformity with degradation studies using radiolabelled material. The interstitial water was separated from the sediment samples by centrifugation. The remaining Sediment was extracted by stirring with a mixture of methanol/N1illipore water (80/20, V N). Test substance concentrations in sediment were analysed from samples with the highest administration rates, i.e. 0.004 mg/L and the lower one, i.e. 0.002 mg/L. The determination of the sediment samples from procedure A was checked by a parallel determination of two recovery samples. These samples gave a recovery rate below 10 %.
- Analysis and calculation of the concentration of the test substance in test water and the sediment compartment (Exposure scenario B): The nominal test concentrations were 0.010, 0.020, 0.040, 0.080, 0.16 and 0.32 mg/kg dry sediment. The actual measured test concentrations of test substance in the water phase were 0.0003, 0.0003, 0.0004, 0.0003. 0.0003 and 0.0004 mg/L at test day 0 (nominal concentrations 0.010, 0.020, 0.040, 0.080. 0.16 and 0.32 mg/kg sediment dry weight). At the end of the test (test day 28) water concentrations were - Effects on the Rate of Emergence, the Development Time and the Development Rate: There were no indications on different sensitivities of sexes, therefore male and female results were pooled for statistical analyses. Calculations of effects on the rate of emergence. the development time and development rate (reciprocal of the development time) were done by using a regression approach. All calculations were based on nominal concentrations in the water phase for exposure scenario A and based on nominal concentrations in the sediment for exposure scenario B. The EC-50 values for emergence rate and development rate of were 0.0041 and >0.004 mg/L for organisms exposed to the test substance via spiking of the water column. The corresponding NOEC values were 0.002 and 0.004 mg/L for emergence rate and development rate, respectively. The EC50 values for emergence rate and development rate of were 0.0815 and > 0.32 mg/kg sediment (dry weight) for organisms exposed to the test substance via spiking of the sediment. The corresponding NOEC values were 0.04 and 0.08 mg/kg for emergence rate and development rate, respectively.
- Effects on the Weight of Larvae: Growth measurements of the larvae via the estimation of the weight were carried out for exposure scenario A and B. Exposure scenario A: After 10 days of exposure 18, 18, 20, 19, 19, 20 and 17 larvae could be found at test concentrations blank, 0.000125, 0.00025, 0.00050, 0.0010, 0.0020 and 0.00040 mg/L. The corresponding average weight of the test groups were 1.24, 1.06, 1.23, 1.19, 1.28, 1.22 and 0.81 mg/larvae. At the highest test concentration the mean weight of the 10 days old larvae was lower than the mean weight of control. Exposure scenario B: After 8 days of exposure 18, 17, 19, 18, 20, 20, 19 and 6 larvae could be found at test concentrations blank, vehicle, 0.010. 0.020, 0.040, 0.080, 0.16 and 0.32 mg/kg sediment (dry weight). The corresponding average weight of the test groups was 0.53, 0.34, 0.52, 0.37, 0.55, 0.57, 0.27 and 0.02 mg/larvae. At the two highest test concentrations the mean weight of the 8 days old larvae was lower than the mean weight of control and vehicle.

Reported statistics and error estimates:

ER and GR are transformed prior to statistical analysis, using an arcsine-squareroot transform. Dunnett tests (α = 5%) for these three parameters are performed to calculate NOEC and LOEC. ECx and 95%—confidence limits are calculated by logit analysis for emergence rate and development rate.

Table 1. Effects of the test substance to the development of C. riparius in 28-days tests

Water-spiked test				Sediment-spiked test
Nominal conc (mg/L)	Mean emergence rate	Mean gender rate, male:female	Mean Development rate	Nominal conc (mg/kg)	Mean emergence rate	Mean gender rate, male:female	Mean Development rate
Control	1.4205	0.7615	0.0642	Control	1.3631	0.8087	0.0643
0.000125	1.0905	0.9027	0.0653	0.01	1.4956	0.7601	0.0668
0.00025	1.5708	0.7865	0.0675	0.02	1.3884	0.7945	0.0664
0.0005	1.1764	0.8507	0.0644	0.04	1.4635	0.7835	0.0627
0.0010	1.4382	0.7731	0.0642	0.08	0.8926 **	1.1445	0.0667
0.0020	1.3776	0.7520	0.0624	0.16	0.0000 ***	--	--
0.0040	0.9226 *	0.7690	0.0631	0.32	0.0000 ***	--	--

*, **, *** statistically significantly different to the control at p < 0.05, p < 0.01, p < 0.001, respectively

Table 2. Average weight of larvae exposed to the test substance

Scenario A: 10 days after exposure			Scenario B: 8 days after exposure
Concentration (mg/L)	Average weight of larvae (mg)	Found Larvae	Concentration (mg/kg)	Average weight of larvae (mg)	Found Larvae
Control	1.24	18	Control	0.53	18
-	-	-	Vehicle	0.34	17
0.000125	1.06	18	0.010	0.52	19
0.00025	1.23	20	0.020	0.37	18
0.00050	1.19	19	0.040	0.55	20
0.0010	1.28	19	0.080	0.57	20
0.0020	1.22	20	0.16	0.27	19
0.0040	0.81	17	0.32	0.02	6

 

Table 3. Summary of effect concentrations

Scenario	Endpoint	EC50	NOEC	LOEC
A (water spk)	Emergence rate	0.0041 mg/L	0.002 mg/L	0.004 mg/L
	Development rate	> 0.004 mg/L	0.004 mg/L	>0.004 mg/L
B (sed. spk)	Emergence rate	0.0815 mg/kg	0.04 mg/kg	0.08 mg/kg
	Development rate	> 0.32 mg/kg	0.08 mg/kg	>0.08 mg/kg

Validation criteria

The validity of these tests are confirmed, because:

- Mortality in the controls < 20 % at the end of the test.

- The emergence in the controls was greater than 80% and the mean development time for the larvae in exposure scenarios A and B

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on results incl. tables'

Conclusions:

In a toxicity test on the sesiment dwelling organism Chironomusriparius, performed in accordance with BBA and OECD guideline, the 28-d NOEC was determined to be 0.04 mg/kg sediment dw. for emergence rate when spiking the sediment, and 0.002 mg/L for emergence rate when spiking the water column.

Executive summary:

In two exposure scenarios, the effects of the test substance on the sediment dwelling organism Chironomus riparius were assessed. The study was conducted according to BBA Guideline Proposal 1995 and OECD Guideline (May 1998), and in compliance with GLP criteria. In scenario A, the test substance was applied to the water column of water-sediment exposure systems, giving nominal exposure concentrations of 0.000125, 0.00025, 0.00050, 0.0010, 0.0020 and 0.004 mg/L. In exposure scenario B, it was the sediment that contained the source of test substance (0.010, 0.020, 0.040 0.080, 0.16 and 0.32 mg/kg sediment dw with a vehicle of acetone) in a water-sediment exposure system. Organisms were exposed for 28 days in 1 L beakers containing about 1.6 cm artificial sediment and approximately 8 cm water at 20 ± 2 ˚C, and a 16 light: 8 dark hours photoperiod (800 – 1000 lux). Each test vessel contained 20 first instar larvae. Control and vehicle control (scenario B only) exposure systems containing no test substance were used for comparison. The biological assessment was based on impacts on full maturation of the larvae to adult midge. Parameters examined were rate and time of emergence and the total number of adult male and female midges. To check the concentrations of the test substance used in the test, the concentrations in the test solutions were determined on days 0, 2, 7, 14, and 28 and 0, 3, 7, 14, and 28 for scenario A and B, respectively. First samples were concentrated using solid phase extraction, followed by reversed phase high performance liquid chromatography. Sediment samples were analysed on study days 0, 7, and 28.

In scenario A (water spk), all water concentrations were <LOD at the end of the test. In scenario B (sed. spk), the mean measured concentrations of the test substance found in the sediment ranged from 116-137% of nominal. The nominal concentrations of the test substance found in the water column in scenario A, and in the sediment in scenario B were used for the calculation and reporting of results. There were no indications on different sensitivities of sexes. After 10 days of exposure, at the highest test concentration, the mean weight of the 10 days old larvae was lower than the mean weight of control in the Exposure scenario A group. In the Exposure scenario B group, after 8 days of exposure, at the two highest test concentrations, the mean weight of the 8 days old larvae was lower. The NOEC values were calculated to be 0.002 and 0.004 mg/L for emergence rate and development rate respectively, for organisms exposed via spiking of the water column. The NOEC values were 0.04 and 0.08 mg/kg for emergence rate and development rate respectively, for organisms exposed via spiking of the sediment.

Description of key information

28-d, NOEC = 0.002 mg/L, Spiking of water column, Chironomus riparius, emergence rate, BBA and OECD guideline, Grate 1999

28-d, NOEC = 0.04 mg/kg sediment dw, spiking of sediment, Chironomus riparius, emergence rate, BBA and OECD guideline, Grade 1999

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

0.04 mg/kg sediment dw

Additional information

One study is available on the toxicity to the sediment dwelling organism Chironomus riparius, following the BBA Guideline Proposal 1995 and OECD Guideline proposal (May 1998), and performed under GLP conditions. This study was selected as the key study. In scenario A, the test substance was applied to the water column of water-sediment exposure systems, giving nominal initial exposure concentrations of 0.000125, 0.00025, 0.00050, 0.0010, 0.0020 and 0.004 mg/L (mean measured concentrations in water column: < LOD (limit of detection), <LOD, <LOD, 0.00026, 0.00044, 0.00049, respectively). In exposure scenario B, it was the sediment that contained the source of test substance (0.010, 0.020, 0.040 0.080, 0.16 and 0.32 mg/kg sediment dw with a vehicle of acetone) in a water-sediment exposure system. Test organisms, Chironomus riparius (20 first instar larvae/test vessel), were exposed to the substance for 28 days, at 20 ± 2 ˚C, and a 16 light: 8 dark hours photoperiod (800 – 1000 lux). Control and vehicle control (scenario B only) exposure systems containing no test substance were used for comparison.

There were no indications on different sensitivities of sexes. After 10 days of exposure, at the highest test concentration, the mean weight of the 10 days old larvae was lower than the mean weight of control in the Exposure scenario A group. In the Exposure scenario B group, after 8 days of exposure, at the two highest test concentrations, the mean weight of the 8 days old larvae was lower. The NOEC values were calculated to be 0.002 and 0.004 mg/L for emergence rate and development rate respectively, for organisms exposed via spiking of the water column. The NOEC values were 0.04 and 0.08 mg/kg sediment dw for emergence rate and development rate respectively, for organisms exposed via spiking of the sediment.