1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

10 Nov 1998 to 22 Apr 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: BBA guideline Proposal

Version / remarks:

Effects of Plant Protection Products on the Development of Sediment-Dwelling larvae of Chironomus riparius in a Water-Sediment System (1995)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for testing of chemicals, Proposal for toxicity test with Chironomidae

Version / remarks:

May 1998

Deviations:

no

Principles of method if other than guideline:

The guideline was a proposed guideline that later turned into OECD 218 and 219.

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 center of the test vessels at day 0, 2, 7, and 28 for expsure scenarios A and B. These samples were analyzed and the concentration of 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 16 and 8.0 mg/L and 400 and 200 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:

DMF for exposure scenairo A and acetone for exposure scenario B

Details on sediment and application:

- Exposure scenario A (Water spike): 3.06 g test substance were mixed and made up to 10 mL with DMF. This solution were diluted with DMF to make solution 2, 3, 4, 5, 6 and 7. Test substance concentrations in this solutions were 306, 153, 76.5, 38.25, 19.13, 9.56 and 4.78 mg/mL DMF. 302 µL of each solution were mixed and made up to 100 mL reconstituted water (M4-medium) to prepare stock solution 1 to 7. The final test solutions were prepared by adding 10 mL (for the highest test concentration 20 mL of stock solution 2) of these stock solutions into the test chamber containing 564 mL M4- medium.
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. Gentle aeration was provided through a glass Pasteur pipette situated about 2 to 3 cm above the sediment.

Exposure scenario B (sediment spike): 2.1117 g test substance were mixed and made up to 10 mL with acetone to prepare SL1. 2.5 and 1.25 mL of this stock solution were mixed and made up to 5.0 mL with acetone to prepare SL 2 and SL 3. 1.25 and 0.625 mL of stock solution 1 were mixed and made up to 10 mL with acetone to SL 4 and SL 5.
After evaporation of the acetone the dry sand was added to the aged sediment (1400 g sediment equivalent to 1046 g dry sediment) to achieve the planned test concentrations and thoroughly mixed with a Mixer for about 30 minutes.

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Culture: 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 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 throughout the culture period.
- Feeding: The larvae are fed with a fish flake food (dry ground powder). 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 test organisms: 1st instar larvae, 2-3 days old
- Handling of the 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 larvae have hatched and are transferred to the test beakers.
- Feeding during test: Commercially available fishfood is used as food. 1 g of the fish food 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:

water and sediment spiked

Hardness:

- Exposure scenario A: 236 - 244 CaCO3/L
- Exposure scenario B: 232 - 260 CaCO3/L

Test temperature:

- Exposure scenario A: 20.6 to 21.4 ˚C
- Exposure scenario B: 19.9 to 21.1 ˚C

pH:

- Exposure scenario A: 7.7 - 9.9
- Exposure scenario B: 7.9 - 8.5

Dissolved oxygen:

- Exposure scenario A: 5.9 - 8.1 mg O2/L
- Exposure scenario B: 7.2 - 8.2 mg O2/L

Conductivity:

- Exposure scenario A: 620 - 665 µS/CM
- Exposure scenario B: 656 - 735 µS/CM

Nominal and measured concentrations:

- Exposure scenario A (water spiked): nominal 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 and 16 mg/L
- Exposure scenario B: nominal 25, 50, 100, 200 and 400 mg/kg dry sediment
Measured concentrations are included in the results section.

Details on test conditions:

TEST SYSTEM
Exposure scenario A
- Test container: 1 L glass beakers with a diameter of 9 cm.
- Sediment amount: a layer of 1-2 cm of artificial sediment (corresponding to 120 g sediment (moist weight))
- Overlying water amount: a height of approximately 8 cm (550 g water).
- Type: Closed (covered with parafilm with small holes to reduce evaporation throughout the test and to allow collection of emerged midges)
- Aeration: 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 test beakers were prepared 8 days before the start of the definitive test (test application) to allow stabilization of the systems under test conditions. The application of the test substance was carried out one day after adding the larvae

Exposure scenario B
- Test container: 1 L glass beakers with a diameter of 9 cm.
- Sediment amount: a layer of 1.6 cm of artificial sediment (sediment spiked with test substance or untreated sediment for control) corresponding to 160 g sediment (moist weight)
- Overlying water amount: a height of approximately 8 cm (550 g water).
- Type: Closed (covered with parafilm with small holes to reduce evaporation throughout the test and to allow collection of emerged midges)
- Aeration: A gentle aeration was carried out for 2.0 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 to 3 cm above the sediment.
- Others: The test beakers were prepared 48 hours before the start of the definitive test (test application) to allow stabilization of the systems under test conditions. Separation of the sediment ingredients when adding the test water. was avoided by covering the sediment with a styropor plate, before pouring the test water (500 g) into the beakers. The plate was carefully removed thereafter.

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

OVERLYING WATER CHARACTERISTICS
M4 medium was used according to Guideline recommendations. The waster column had a depth of about 8 cm (at the end of exposure 6 cm).
- Water quality measurements: The water temperature was continuously measured in the test room and in a test vessel at different days. Oxygen content and pH were measured once weekly with appropriate electrodes directly in the test vessels. The water conductivity and the water hardness were measured after preparing the M4-medium.

CHARACTERIZATION OF ARTIFICIAL SEDIMENT (Exposure scenario A & B)
- 5.5% peat
- 20% kaolin clay
- 74.5^% industrial sand (fine sand with more than 50% of the particles between 50 and 200 microns)
- the pH was adjusted to 6.7 (exposure scenario A) and 6.8 (exposure scenario B) by adding chemically pure calcium carbonate
- the content of organic carbon was determined to be about 1.7%
The sediment layer was about 1.6 cm thick

OTHER TEST CONDITIONS (Exposure scenario A & B)
- Photoperiod: light:dark-rhythm (16-8h) with 30 minutes transition period
- Light intensity: 800-1000 lux

EFFECT PARAMETERS MEASURED (Exposure scenario A & B)
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 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 9 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.

Biological Endpoints
The following parameters were measured and considered relevant endpoints:
- rate of emergence, i.e. the sum of midges emerged per replicate divided by the number of larvae introduced
- the mean development time. i.e. the mean time between the application of the test substance (day 0) and the emergence of adults
- The rate of development, i.e. the portion of larval development per day which is calculated from the reciprocal of development time.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: sediment spiking

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: sediment spiking

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: water spiking

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: water spiking

Details on results:

An overview of the results is provided in Table 1A and 1B in 'Any other information on results. incl. tables'
ANALYTICAL RESULTS
- Exposure scenario A: The nominal test concentrations were 0.25. 0.50, 1.0, 2.0. 4.0, 8.0 and 16 mg/L. The actual measured test concentrations in the water phase were 0.09, 0.54, 1.2. 2.3, 7.9 and 16.5 mg/L at test day 0 (1-3 hours after application). At the end of the test (test day 28) water concentrations were < LOD (limit of quantification), < LOD, 0.16, 0.40. 0.94, 2.0 and mg/L. Test substance concentrations in sediment were analysed from samples with the highest
administration rates, i.e. 16 mg/L and the lower one. i.e. 8.0 mg/L. At day 0,7 and 28 the measured test substance concentrations in the sediment (incl. interstitial water) were 1.39, 24.6 and 30.80 mg/kg sediment (wet) at a nominal concentration 16 mg/L. At a nominal concentration of 8.0 mg/L, the sum of determined concentrations of the test substance were 1.13. 12.1 and 14.7 mg/kg sediment at day 0. 7 and 28. respectively. Total recovery from test system at a nominal concentration of 16 mg/L were 106. 92 and 72% of the nominal concentration at day 0. 7 and 28 equivalent to the measured concentration of the test substance of 9.70, 8.43 and 6.63 mg/vessel (calculated with the sum of determined content of the test substance). The corresponding figures for a nominal concentration of 8.0 mg/L were 4.69, 4.03, and 3.01 mg/vessel equivalent to 102, 88 and 66% of the nominal concentration at day 0, 7 and 28.
- Exposure scenario B: The nominal test concentrations were 25. 50, 100. 200 and 400 mg/kg dry sediment. The actual measured test concentrations of the test substance in the water phase were 0.12, 0.53, 0.96 2.55 and 5.93 mg/L at test day 0. At the end of the test (test day 28) water concentrations were 0.40, 1.05, 2.12, 6.47 and 17.23 mg/L. Test substance concentrations in sediment were analysed from samples with the highest administration rates, i.e. 400 mg/kg and the lower one, i.e.200 mg/kg. At day 0, 7 and 28 the measured test substance concentrations in the sediment (incl. interstitial water) were 275.8, 254.1 and 237.6 mg/kg sediment (wet) at a nominal concentration of 400 mg/kg sediment (dry weight) equivalent to 299 mg/kg sediment (wet). At a nominal concentration of 200 mg/kg sediment (dry weight) equivalent to 149.5 mg/kg sediment (wet) the sum of determined concentrations of the test substance were 139.4, 126.4 and 118.1 mg/kg sediment (wet) at day 0, 7 and 28, respectively. Total recovery from test system at a nominal concentration of 400 mg/kg (48.1 were 93, 87 and 84 % of the nominal concentration at day 0, 7 and 28 equivalent to the measured concentrations of the test substance of 44.9, 42.1 and 40.2 mg/vessel (calculated with the sum of determined content of the test substance. The corresponding figures for a nominal concentration of 200 mg/kg (24.05 mg/vessel) were 22.95, 21.48 and 20.50 mg/vessel equivalent to 95, 89 and 85 % of the nominal concentration at day 0. and 28.

BIOLOGICAL EFFECTS
- Observations: 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.
In exposure scenario A, the NOEC values for emergence rate and development rate were determined to be 8.0 and 4.0 mg/L, respectively.
In exposure scenario B, the NOEC values for emergence rate and development rate were determined to be 25 and 50 mg/L, respectively.

- Effects on the Weight of Larvae:
Exposure scenario A: After 10 days of exposure, 18, 19, 20, 19, 19, 20, 20, 8 and 14 larvae could be found at blank, vehicle control and test concentrations of 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 and 16 mg/L. The corresponding average weight of the test groups were 1.02, 1.05, 0.45, 0.71, 0.53, 0.62, 0.72, 0.17 and 0.51 mg/larvae. A clear influence to the size of the larvae could be seen at test concentrations of 8.0 and 16 mg/L.
Exposure scenario B: After 9 days of exposure 7, 13, 13, 13, 15, 15 and 0 larvae could be found in blank. vehicle control and test concentrations of 25, 50, 100, 200 and 400 mg/kg sediment (dry weight). The corresponding average weight of the test groups was 0.29, 0.4, 0.33, 0.29, 0.37 and 0.23 mg/larvae. No clear influence could be seen at all test groups where larvae could be found.

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 1A. Sum of larvae, rate emergence (ER), gender rate (GR), and development rate (X)_ Exposure scenario A

obs	Group	Day	NE	NMALE	ER	GR	X	mg/L
1	b	28	17	11	0.80952	0.64705	0.069885	0.00
2	b	28	20	8	1.00000	0.40000	0.070086	0.00
3		28	20	12	1.00000	0.60000	0.067724	0.00
4	v	28	20	8	1.00000	0.40000	0.068072	0.00
5	v	28	19	8	0.95000	0.42105	0.068992	0.00
6	v	28	17	9	0.85000	0.52941	0.068297	0.00
7	0.25	28	20	9	1.00000	0.45000	0.067975	0.25
B	0.25	28	17	9	0.85000	0.52941	0.069323	0.25
9	0.25	28	16	12	0.80000	0.75000	0.074533	0.25
10	0.5	28	17	10	0.85000	0.58823	0.070616	0.50
11	0.5	28	20	11	1.00000	0.55000	0.069310	0.50
12	0.5	28	19	7	0.95000	0.36842	0.064365	0.50
13	1.0	28	20	12	1.00000	0.60000	0.071075	1.00
14	1.0	28	19	8	0.95000	0.42105	0.067658	1.00
15	1.0	28	16	8	0.80000	0.50000	0.067766	1.00
16	2.0	28	15	9	0.75000	0.60000	0.070416	2.00
17	2.0	28	19	9	0.95000	0.47368	0.068513	2.00
18	2.0	28	19	10	0.95000	0.52631	0.068782	2.00
19	4.0	28	20	5	1.00000	0.25000	0.064151	4.00
20	4.0	28	18	9	0.90000	0.50000	0.065378	4.00
21	4.0	28	20	11	1.00000	0.55000	0.068418	4.00
22	8.0	28	20	11	1.00000	0.55000	0.061565	8.00
23	8.0	28	17	11	0.85000	0.64705	0.065729	8.00
24	8.0	28	17	6	0.85000	0.35294	0.058805	8.00
25	16.0	28	0	0	0.00000	-	-	16.00
26	16.0	28	0	0	0.00000	-	-	16.00
27	16.0	28	0	0	0.00000	-	-	16.00

NE:  Sum of midges emerged per vessel

ER: Emergence Rate

GR: Gender rate

X: Mean of development rate per vessel

 

Table 1B. Table 1 Sum of larvae, rate emergence (ER), gender rate (GR), and development rate (X)_ Exposure scenario B

obs	Group	Day	NE	NMALE	ER	GR	X	mg/kg
a	b	28	19	15	0.95000	0.70947	0.064215	0
2	b	28	20	11	1.00000	0.55000	0.063836	0
3	b	28	17	13	0.85000	0.76471	0.062987	0
4	v	28	19	11	0.95000	0.57895	0.062177	0
5	v	28	17	7	0.85000	0.41176	0.062177	0
6	v	28	20	11	1.00000	0.55000	0.062026	0
7	25	28	16	6	0.80000	0.37500	0.058595	25
	25	28	17	8	0.85000	0.47059	0.060501	25
9	25	28	15	9	0.71429	0.60000	0.055933	25
10	50.0	28	14	6	0.70000	0.42857	0.057687	50
11	50.0	28	16	10	0.80000	0.62500	0.060528	50
12	50.0	28	14	6	0.70000	0.42857	0.061680	50
13	100.0	28	15	6	0.75000	0.40000	0.057697	100
14	100.0	28	15	9	0.75000	0.60000	0.058053	100
15	100.0	28	10	6	0.50000	0.60000	0.057576	100
16	200.0	28	1	1	0.05000	1.00000	0.051282	200
17	400.0	28	0	0	0.00000	-	-	200
18	400.0	28	0	0	0.00000	-	-	400
19	400.0	28	0	0	0.00000	-	-	400
20	400.0	28	0	0	0.00000	-	-	400

 

Validity of the test
Since emergence in the controls was greater than 80% and the mean development time for the larvae in exposure scenarios A and B was not later than 23 days after application, the validity of these tests is confirmed.

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on results incl. tables'

Conclusions:

In a toxicity test on the sediment dwelling organism Chironomus riparius, performed in accordance with BBA and OECD guideline, the 28-d NOEC values were determined to be 25 and 50 mg/kg sediment dw. for emergence rate and development rate, respectively, when spiking the sediment. The 28-d NOEC values were 8.0 and 4.0 mg/L for emergence rate and development rate, respectively, when spiking the water column.

Executive summary:

The effects of the test substance on the sediment-dwelling organism Chironomus riparius were assessed using two exposure scenarios. The 28-d study was conducted according to BBA Guideline Proposal 1995 and OECD Guideline proposal (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 0.25, 0.50, 1.0, 2.0, 40, 8.0 and 16 mg/L. In exposure scenario B, it was the sediment that contained the source of test substance (25, 50, 100, 200 and 400 mg/kg sediment dw) in a water-sediment exposure system. Organisms were exposed for 28 days in 1 L beakers containing about 1.5 cm artificial sediment and approximately 8 cm water at 20 ± 2 ˚C, and a 16 light: 8 dark hours photoperiod with 30 mins transition period (800 – 1000 lux). Each test vessel contained 20 first instar larvae. Control and vehicle control exposure systems (DMF for scenario A and acetone for scenario B) containing no test substance were used for comparison. The biological assessment was based on impacts on the full maturation of the larvae to adult midge. The main parameters examined were the rate and time of emergence and the total number of adult male and female midges. The concentrations of the test substance in the test solutions were determined on days 0, 2, 7, 14, and 28 for scenarios A and B. Sediment samples were collected on Day 0, 7 and 28 from the 8 and 16 mg/L treatment groups in scenario A, and  200 and 400 mg/kg sediment dw treatment groups in scenario B. The collected samples were extracted and analysed in HPLC with UV detection.

In scenario A (water spiked), the actual measured test concentrations in the water phase were 0.09, 0.54, 1.2, 2.3, 4.5, 7.9 and 165 mg/L at Day 0. At the end of the test (test day 28) water concentrations were <LOD, <LOD. 0.16, 0.40, 0.94, 2.0 and 4.7 mg/L. There were no indications of different sensitivities of sexes. After 10 days of exposure, 18, 19, 20, 19, 19, 20, 20, 8 and 14 larvae could be found at the blank, vehicle control and test concentrations of 0.25, 0.50, 1.0,  2.0, 4.0, 8.0 and 16 mg/L respectively. The corresponding average weight of the test groups were 1.02, 1.05, 0.45, 0.71, 0.53, 0.62, 0.72, 0.17 and 0.51 mg/larvae. A clear influence on the size of the larvae could be seen at test concentrations of 8.0 and 16 mg/L.

In scenario B (sediment spiked), the mean measured concentrations of the test substance found in the sediment ranged from 84-95% nominal. After 9 days of exposure, 7, 13, 13, 13, 15, 15 and 0 larvae could be found in the blank, vehicle control and test concentrations of 25, 50, 100, 200 and 400 mg/kg sediment (dry weight), respectively. The corresponding average weight of the test groups was 0.29, 0.4, 0.33, 0.29, 0.37 and 0.23 mg/larvae. No clear influence could be seen in all test groups where larvae could be found.

The 28-d NOEC values were calculated to be 8.0 and 4.0 mg/L for emergence rate and development rate, respectively, for organisms exposed via spiking of the water column. The NOEC values were 25 and 50 mg/kg for emergence and development rates, respectively, for organisms exposed via sediment spiking.

Description of key information

28-d, NOEC = 25 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:

25 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. Two exposure scenarios were included.

In scenario A, the test substance was applied to the water column of water-sediment exposure systems, giving nominal initial exposure concentrations of 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 and 16 mg/L (mean measured concentrations in the water column at the end of the test: < LOD, < LOD, 0.16, 0.40, 0.94, 2.0 and 4.7 mg/L, respectively). In exposure scenario B, it was the sediment that contained the source of the test substance (25, 50, 100, 200 and 400 mg/kg sediment dw) 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 with 30 mins transition period (800 – 1000 lux). Control and vehicle control (DMF for scenario A and acetone for Scenario B) exposure systems containing no test substance were used for comparison.

There were no indications of different sensitivities of sexes. After 10 days of exposure, a clear influence on the size of the larvae could be seen at test concentrations of 8.0 and 16 mg/L in exposure scenario A. In exposure scenario B, after 9 days of exposure, No clear influence could be seen at all test groups where larvae were found. The 28-d NOEC values were calculated to be 8.0 and 4.0 mg/L for emergence rate and development rate, respectively, for organisms exposed via spiking of the water column. The NOEC values were 25 and 50 mg/kg for emergence and development rates, respectively, for organisms exposed via sediment spiking.