Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

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:

23 Oct 2001 to 27 Feb 2002

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:

February 2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1790 (Chironomid Sediment Toxicity Test)

Version / remarks:

April 1996

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Specimens of an appropriate volume of the aqueous test solution, i.e. 25 mL specimens from 2 beakers per concentration and control were taken from the centre of the test vessels at day 0, 2, 7, 14 and at the end of exposure and analysed for the concentration of the test item. If samples were not analysed immediately, they were frozen (<-18°C).
At the start, after 7 days and the end of the experiment, the concentration of the test item in the sediment was measured only for the control, the highest test concentration and the lower one, i.e. 1.5 and 3.0 µg/L. 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. Test organisms were added to these chambers (at the beginning of the test and for 7 days sampling).

Vehicle:

yes

Remarks:

Dimethylformamide (DMF)

Details on sediment and application:

PREPARATION OF STOCK SOLUTION AND FINAL TEST SOLUTIONS:
50.00 mg of test item was weighed in and made up to 10 mL with DMF. 300 µL of this stock solution were added to approximately 900 mL of M4 water, treated with ultrasound for 10 minutes and then made up to 1000 mL with M4
water in a volumetric flask (stock solution). 116 mL of the stock solution were mixed and made up to 1000 mL with M4 water to prepare application solution 1. 58, 29, 14.5 and 7.25 mL of the stock solution were mixed and made up to 1000 mL M4 water to prepare application solutions 2, 3, 4 and 5, respectively. 10.0 mL of application solutions 5, 4, 3, 2 and 1 were added into the test chamber containing 580 mL M4-medium.to prepare test solutions with concentrations of 0.188, 0.375, 0.75, 1.5 and 3.0 µg test item / L, respectively.

PREPARATION OF SPIKED WATER
- Details of spiking: The application of the test item was carried out one day after adding the larvae. The application solutions were added to the water column of the test vessels and the upper water layer was gently mixed with a pipette to ensure homogeneous distribution without disturbing the sediment. The test began after application of the test item.
- Controls: For the blank controls 10.0 mL of M4 water not containing any test item were added into each test chamber to prepare the blank test solutions. For the vehicle control an application solution was made up with 35 DMF in 1000 mL M4 water, of this solution 10.0 mL was added to each vehicle control test chamber.
- Chemical name of vehicle: Dimethylformamide

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Age of parental stock: 2 - 3 days old
- Handling of egg masses and larvae: The first 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:
Food type: commercially available fish food
Amount: 1 mg per larvae

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

25 d

Exposure phase:

total exposure duration

Hardness:

249 mg CaCO3/L

Test temperature:

20 ± 2 °C

pH:

6.0 - 9.0

Nominal and measured concentrations:

Nominal concentrations: 0.188, 0.375, 0.75, 1.5 and 3.0 µg/ L

Details on test conditions:

TEST SYSTEM
- Test container: 1 L glass beakers measuring 9 cm in diameter.
- Aeration: yes; Gentle aeration was provided through a glass Pasteur Pipette situated about 2 to 3 cm above the sediment layer.
- Depth of sediment and overlying water: The vessels contained 1 to 2 cm of artificial sediment and a water column 7 to 9 cm deep.

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: 1 mg food per day per larvae during the course of the experiment
- Type and preparation of food: A commercially available fish food is used as food. 2 g food is ground finely and suspended in 40 mL water

OVERLYING WATER CHARACTERISTCS
- Type of water: M4 water, reconstituted, bi-distilled water

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5.5
- Particle size distribution : >50% of sand particles are 50 to 200 µm
- % sand: 74.5 (Industrial sand)
- % clay: 20
- 1.3% calcium carbonate was used to reach a pH of 7.4;
- content of organic carbon was determined to be 2.2 %.

OTHER TEST CONDITIONS
- Light quality: Fluorescent light
- Photoperiod: 16 hours daily with 30 min. transition period, 8 hour darkness.
- Light intensity: Light intensity of 760 Lux (mean of 9 measurements).

EFFECT PARAMETERS MEASURED:
During the period of emergence (day 11 till day 22, day 25) a daily check of emerged midges was carried out. The sex and number of adults emerged was recorded. Afterwards adults were removed from the test vessels. Visual assessments of any behavioural differences or other noticeable changes compared with the control were made at test days 6, 7, 11, 12, 14, 15, 16, 17, 18, 20 and 21. For an estimation of larval survival and growth at day 11 additional vessels were set up at the start. The sediment from these additional vessels was sieved using a 250 µm sieve to retain the larvae. Criteria for death were immobility and/or lack of reaction to a mechanical stimulus. The dry weight of the surviving larvae per test vessel was measured after drying at 60°C to a constant weight. The mean individual dry weight per vessel was calculated.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Test concentrations: 0.188, 0.375, 0.75, 1.5 and 3.0 µg test item/L

Reference substance (positive control):

no

Key result

Duration:

25 d

Dose descriptor:

NOEC

Effect conc.:

0.75 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Key result

Duration:

25 d

Dose descriptor:

EC50

Effect conc.:

1.07 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Duration:

25 d

Dose descriptor:

NOEC

Effect conc.:

1.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Duration:

25 d

Dose descriptor:

EC50

Effect conc.:

2.69 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Duration:

25 d

Dose descriptor:

NOEC

Effect conc.:

0.188 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Sublethal effects

Details on results:

Analytical results
The nominal concentrations of the test substance which were spiked into the water column of the test vessels were 0.188, 0.375, 0.75, 1.5 and 3.0 test item µg/L. Measured concentrations of the test substance in the water column were close to nominal concentrations at the beginning of the test, in the range of 99 to 118%. After two days of the test measured concentrations of the test substance had fallen to below the detection limit of <0.2 µg/L, except at the highest test concentration of 3.0 test item µg/L where 11% of the nominal concentration was measured in samples taken from the water column. At 7 days of the study, measured concentrations of the test substance at all treatment levels were below the detection limit of <0.2 test item µg/L. Since measured concentrations of the test substance in water samples taken at 7 days of the test were below the detection limit, water specimens taken at 14 days and test end were not analysed anymore.
Concentrations of the test substance in the sediment and the interstitial water were measured only in the two highest test concentrations, i.e. 1.5 and 3.0 µg/L and in the control. Measured concentrations of the test substance in the sediment and the interstitial water were below the detection limit at the beginning of the test, after 7 days of the test and the end of the test.

Gender ratio
There were no indications on different sensitivities of sexes at treatments of 0.188, 0.375 and 0.75 µg/L (the number of emerged midges at concentrations of 1.5 and 3.0 µg/L were too low and hence were excluded from statistical analysis).

Emergence of midges
The mean emergence for the control, vehicle and test concentrations of 0.188, 0.375, 0.75, 1.5 and 3.0 µg/L was 87.5, 80.0, 76.3, 60.0, 26.3 and 7.5%, respectively. For the calculation of ECx-values for emergence, data was corrected for control ‘mortality' before regression analysis was carried out. Midges not emerged were considered as dead.

Emergence ratio
The highest concentration of the test substance without significant effect on emergence ratio of midges was determined to be a test concentration of 0.75 µg/L (NOEC emergence ratio). The LOEC (emergence ratio) was determined to be a test concentration of 1.5 µg/L test substance.

Development rate
The highest concentration of the test substance without significant effect on development rate of midges was determined to be a test concentration of 1.5 µg/L (NOECdevelopment rate). The LOEC (development rate) was determined to be a test concentration of 3.0 µg/L test substance.

Effects on the weight of larvae
After 11 days of exposure 18, 17, 14, 17, 20, 18, and 11 larvae were found in the blank control, vehicle and at test concentrations of 0.188, 0.375, 0.75, 1.5 and 3.0 test item µg/L, respectively. The corresponding average dry weights of the test groups were 1.28, 1.26, 1.01, 1.08, 1.27, 1.20 and 1.20 mg/larva. In the blank control, the vehicle control and a test concentration of 0.375 test item µg/L, one pupae each in the control treatments and three pupae at the test item concentration were found. Pupae were excluded from estimations of average weight of larvae. There are no indications that there is a concentration dependent decrease of average weight of larvae at the tested treatment levels. Since only one replicate per control, vehicle and test concentration was performed, no statistical analysis was carried out.

Sublethal effects and other observations
Visual assessments of any behavioural differences or other noticeable changes compared with the control were made at test days 6, 7, 11, 12, 14, 15 ,16, 17, 18, 20 and 21 days after application of the test item. On day 8 a slight fungal growth was observed on all test vessels hence only half the portion of food solution was fed. On day 7 of the test, galleries on the sediment surface were observed. On day 11 it was observed that the overlying water of the test glasses was slightly cloudy.
- Observations in blank control: On day 12 one dead larva was visible on the sediment surface. On day 18 one dead pupa was recorded.
- Observations in vehicle control: On day 16 and 17 one half-emerged midge each was recorded.
- Observations in test 0.188 µg/L: On day 12 one dead larva was recorded on the sediment surface. On day 17 two dead pupae were recorded. On day 18 two dead white larva were visible on the sediment surface.
- Observations in test 0.375 µg/L: On day 14 two midges with elongated greenish bodies and deformed wings, one dead pupa on water surface and two dead larvae on sediment surface were recorded. On day 18 one dead larva and 4 dead pupae on sediment surface were observed.
- Observations in test 0.75 µg/L: On day 14 one dead pupa on water and a dead male and female midge with greenish bodies on water surface were observed. On day 15 three half-emerged midges on water-surface and one dead male midge were recorded. On day 16 one half-emerged midge, one dead larva and four dead pupae were visible on sediment surface. On day 18 one half-emerged midge was recorded.
- Observations in test concentration 1.5 µg/L: On day 12 one dead larva was visible. On day 14 at this treatment level 17 pupae with contracting bodies were observed on the sediment surface as well as 2 dead larvae and 2 dead pupae. Also 3 midges with elongated greenish bodies were recorded. On day 15 five female midges with reddish bodies and one male midge with greenish elongated body were observed. On day 16 three dead larvae and four dead pupae were recorded on the sediment surface. On day 17 one female midge with elongated reddish body and one dead pupa was recorded. On day 18 at this treatment level 26 dead pupae, 6 dead larvae and one half-emerged midge were visible.
- Observations in test concentration 3.0 µg/L: On day 12 at this treatment level 8 pupae with contracting bodies were observed on the sediment surface as well as 1 dead pupa. On day 14, 20 pupae with contracting bodies were observed on the sediment surface as well as 1 dead larva and 2 dead pupae. Also 1 female midge with a reddish body was recorded. On day 15 three half-emerged midges were observed on the water surface. On day 17 one midge with deformed wings was recorded. On day 18, 35 dead pupae and 3 dead larvae were visible on the sediment surface.

Reported statistics and error estimates:

Emergence ratio (ER) and growth ratio (GR) were transformed prior to statistical analysis, using an arcsine-square root transformation. Values were transformed using Microsoft Excel worksheets. Dunnett multiple sequential test procedures were performed = 0.05%, one-sided, smaller) for the parameters GR, ER and development rate to calculate NOEC and LOEC values. The statistical program used for these analyses was "EASY ASSAY Multiple Testing" (Program by Ratte H T, 1995a; Spirit Software, D-52477 Alsdorf). ECx and 95%-confidence limits for emergence and development rate were calculated by probit analysis using the statistical program “EASY ASSAY Critical values" (Program by Ratte H T, 1995b; Spirit Software, D--52477 Alsdorf).

Table 10. Summary of development rates

Concentration (ug/L)	mean development rate per vessel (1Iday)	mean development rate per vessel (1Iday)	mean development rate per vessel (1Iday)	mean development rate per vessel (1Iday)	mean development rate per treatment	% of control	% inhibition
	Replicate A	Replicate B	Replicate C	Replicate D
Control	0.0683	0.0711	0.0678	0.0742	0.0704	100	0.00
Vehicle	0.0704	0.0705	0.0692	0.0683	0.0696	98.94	1.06
0.188	0.0678	0.0637	0.0702	0.0714	0.0683	97.07	2.93
0.375	0.0689	0.0890	0.0647	0.0649	0.0669	95.04	4.96
0.75	0.0685	0.0697	0.0649	0.0705	0.0684	97.20	2.80
1.5	0	0.0589	0.0637	0.0715	0.0485	68.97	31.03
3.0	0	0.0662	0.0500	0	0.0295	41.99	58.01

 

Table 11. Average weight of larvae (11 days after start of exposure)

Concentration (ug/L)	Average weight of larvae (mg)	Larvae Found	Pupae Found*
Control	1.275	18	1
Vehicle	1.261	17	1
0.188	1.007	14	0
0.375	1.078	17	3
0.75	1.272	20	0
1.5	1.204	18	0
3.0	1.200	11	0

 * pupae were excluded from estimations of average weight of larvae

 

Table 15. Analytical determinations of the test substance in water samples

Nominal concentration of test substance (ug/L)	Determined concentrations of test substance	Determined concentrations of test substance	Determined concentrations of test substance	Determined concentrations of test substance.	Determined concentrations of test substance	Determined concentrations of test substance
	Initial (0h)	Initial (0h)	2 days	2 days	7 days	7 days
	(ug/L)	%	(ug/L)	%	(ug/L)	%
Blank	<0.2	-	<0.2	-	<0.2	-
Vehicle	<0.2	-	<0.2	-	<0.2	-
0.188	0.221	118	<0.2	-	<0.2	-
0.375	0.401	107	<0.2	-	<0.2	-
0.75	0.756	101	<0.2	-	<0.2	-
1.5	1.57	105	<0.2	-	<0.2	-
3.0	2.97	99	0.32	11	<0.2	-
Mean		106
Min.		99
Max.		118		11

 Values were corrected for recovery (mean recovery = 97%;

Values were calculated using a Microsoft Excel worksheet, hence calculations are carried out using unrounded values.

  

Table 16. Analytical determinations of the test substance in interstitial water

Nominal concentration of test substance (ug/L)	Initial (0h)	Initial (0h)	7 days	7 days	25 days (End)	25 days (End)
	Vol. of interst. water (ml)	Found conc. of test substance	Vol. of interst. water (ml)	Found conc. of test substance	Vol. of interst. water (ml)	Found conc. of test substance
Blank	14.5	<0.7 ug/L	20.5	<0.5 ug/L	34.5	<0.3 ug/L
1.5	12.7	<0.8 ug/L	17.5	<0.6 ug/L	33.0	<0.3 ug/L
3.0	18.0	<0.6 ug/L	19.5	<0.5 ug/L	28.5	<0.4 ug/L

 

 

Table 17. Analytical determinations of the test substance in sediment at start.

 Limit of quantification (LOQ): 0.010 ug/g wet sediment = 1.8 ug / 100 g dry sediment

Sediment sample	Dry content of sediment	test substance found (ug/g)	test substance calc. on dry content
Blank	55.5%	< 0.01	< 1.8
1.5 ug/Liter	54.7%	< 0.01	< 1.8
3.0 ug/Liter	55.4%	< 0.01	< 1.8

 

 Table 19. Analytical determinations of the test substance in sediment at test end

 To improve the limit of quantification, all samples have been spiked with 0.205 ug test substance. Limit of quantication (LOQ): 0.005ug lg wet sediment = 0.9ug / 100 g dry sediment

Sediment sample	Dry content of sediment	test substancefound (ug/g)	test substancecalc. on dry content
Blank	62.0%	^1)	^1)
1.5 ug/Liter	60.7%	< 0.005	< 0.9
3.0 ug/Liter	55.5%	< 0.005	< 0.9

 ¹⁾This sample has been used as a “blind"-sample, that means the peak area oftest substancefound in this sample is a blind-peak and will be subtracted from thetest substancepeak in the samples 1.5 ug/Liter and 3.0 ug/Liter.

There are enough results for Blank-samples, to assume there is < 0.9 ug test substance/ g dry sediment, or < 1.5 ug/Liter in the aqueous phase.

Validity criteria fulfilled:

yes

Conclusions:

In a sediment-water toxicity study in the sediment dwelling organism Chironomus riparius, performed in accordance with OECD TG 219, the highest concentration of the test substance without significant effect on emergence ratio of midges was determined to be a test concentration of 0.75 µg/L (NOEC emergence ratio). The LOEC (emergence ratio) was determined to be a test concentration of 1.5 µg/L test substance. The highest concentration of the test substance without significant effect on development rate of midges was determined to be a test concentration of 1.5 µg/L (NOEC development rate). The LOEC (development rate) was determined to be a test concentration of 3.0 µg/L test substance. The overall observed NOEC of the test substance in this study based on biological observations made during the study was determined to be a concentration of 0.188 µg/L and the overall ‘observed’ LOEC was determined to be at a treatment level of 0.375 µg/L.

Executive summary:

The chronic toxicity of the test substance toChironomus riparius (2-3 days old, first instar larvae) was assessed in a 28-day water/sediment system under static conditions. The study was performed in accordance with the proposal for OECD guideline TG 219 and under GLP conditions. Nominal test concentrations were 0.188, 0.375, 0.75, 1.5 and 3.0 µg/L, with untreated and solvent control. The test was performed in 1 L glass beakers, with four replicates per treatment each containing 20 midge larvae and approximately 1-2 cm of sediment (artificial OECD soil) and approximately 8 cm of overlying water. Additional replicates were set up for analytical measurements on sediment and pore water on days 1 and 7. Midge larvae were added to the test system one day prior to the addition of test substance. Treatment was performed by spiking the water with an aliquot (10 mL) of a stock solution of the test substance in test water containing 0.03% DMF.

On the day of application the analytically determined concentrations of the test substance in the overlying water represented 99-118% of nominal. On day 2 the measured concentrations had declined below the limit of detection (<0.2 μg/L) at all but the highest concentration (11% of nominal). After 7 days of exposure, all measured concentrations had fallen below the limit of detection. Actual concentrations in sediment and pore water at the two highest concentrations were below the limits of detection throughout the study. Water quality parameters were in accordance with the OECD 219 guideline with a minor deviation in temperature at the start of the test, which did not affect the study result. Results showed that the emergence rate was statistically significantly decreased at the highest two concentrations, while development rate was statistically significantly reduced at the highest concentration. The resulting EC50-values were based on nominal concentrations (analytically confirmed for initial concentrations) and determined to be 1.07 µg/L for emergence and 2.69 μg/L for development. The NOEC for emergence and development rate were 0.75 and 1.5 μg/L, respectively.

Description of key information

28 -d NOEC = 0.75 μg/L, Chironomus riparius, emergence, OECD TG 219, Pfeifle 2002

28 -d NOEC = 1.5 μg/L, Chironomus riparius, development rate, OECD TG 219, Pfeifle 2002

Additional information

OECD TG 210, Pfeifle 2002

The chronic toxicity of the test substance to Chironomus riparius (2-3 days old, first instar larvae) was assessed in a 28-day water/sediment system under static conditions. The study was performed in accordance with the proposal for OECD guideline TG 219 and under GLP conditions. Nominal test concentrations were 0.188, 0.375, 0.75, 1.5 and 3.0 µg/L, with untreated and solvent control. The test was performed in 1 L glass beakers, with four replicates per treatment each containing 20 midge larvae and approximately 1-2 cm of sediment (artificial OECD soil) and approximately 8 cm of overlying water ('M4' medium). Additional replicates were set up for analytical measurements on sediment and pore water on days 1 and 7. Midge larvae were added to the test system one day prior to the addition of test substance. Treatment was performed by spiking the water with an aliquot (10 mL) of a stock solution of the test substance in test water containing 0.03% DMF.

On the day of application the analytically determined concentrations of the test substance in the overlying water represented 99-118% of nominal. On day 2 the measured concentrations had declined below the limit of detection (<0.2 μg/L) at all but the highest concentration (11% of nominal). After 7 days of exposure, all measured concentrations had fallen below the limit of detection. Actual concentrations in sediment and pore water at the two highest concentrations were below the limits of detection throughout the study. Water quality parameters were in accordance with the OECD 219 guideline with a minor deviation in temperature at the start of the test (17.8°C instead of minimum 18°C), which did not affect the study result. Results showed that the emergence rate was statistically significantly decreased at the highest two concentrations, while development rate was statistically significantly reduced at the highest concentration. The resulting EC50-values were based on nominal concentrations (analytically confirmed for initial concentrations) and determined to be 1.07 µg/L for emergence and 2.69 μg/L for development. The NOEC for emergence and development rate were 0.75 and 1.5 μg/L, respectively.