N-alkylester N-substituted heterocyclic diazo aryl amine

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:

2017-07-26 to 2017-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 233 (Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment)

Version / remarks:

2010

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Phase 1 and 3: The concentrations of the test item were analytically verified via LC-MS-MS on days 0, 7 and 28. The test item was analysed in the sediment layer, pore water and overlying water. Two additional replicates were set up for analytical verification on day 0 and day 7. On day 28, one replicate of the biological part was analysed per concentration.
Phase 2: The concentrations of the test item were analytically verified after an equilibration time of 7 days in the water and sediment layer of the crystallising dish.

Vehicle:

yes

Remarks:

Tetrahydrofuran (THF)

Details on sediment and application:

1. Preparation of the test solutions: For the test solution with a saturation of 100% the saturated solution was used without further dilution. The nominal concentration is 6.00 g/L. For the test solutions with saturations of 50 and 25% the saturated solution was correspondingly diluted with THF.

2. Application: Due to the very low solubility of the test item in water, the application of the test item was carried out into the sediment. The spiking procedure of the sediment was carried out as specified below. The study was carried out with 10 replicates. The test item is not completely soluble in Tetrahydrofuran (THF) at the required concentration level for the maximum application concentration (1000 mg/kg dry weight) recommended by the guideline OECD 233. The solubility in organic solvents was checked in the preliminary range finding test and THF was determined as the best solvent for application. Thus, the test item was weighed and dispersed with Tetrahydrofuran (THF) to prepare the stock solutions. The stock solution was stirred with a magnetic stirrer for 24 ± 1 hours at room temperature (20 ± 2°C) and, after a brief settlement phase from 30 – 45 minutes filtrated through a cellulose filter paper (Macherey - Nagel, MN 615 ¼ diameter 320 mm). Since undissolved material was visible in the stock solution, which could not be removed by settlement but might have led to disturbances and uneven distribution of the test item in the sediment, the filtration step could not be avoided. The test item was weighed based on a calculation with 12 test replicates (10 replicates were prepared per test group as specified below). For spiking of the sediment 240 g quartz sand per test group (20 g quartz sand per replicate) was prepared. Since on study day -2, a test item analysis from the treated quartz sand was carried out, additional 40 g quartz sand was prepared per test item concentration, resulting in a total amount of 280 g quartz sand to be prepared (50 mL spiking solution per 280 g quartz sand). For spiking the sediment of the test item concentration and the solvent control, 50 mL of the corresponding spiking solution and solvent was transferred to 280 g quartz sand. The dispersed test item was thoroughly mixed with the quartz sand with a spatula. Afterwards, the solvent was evaporated for 1.55 - 2.05 hours (phase 1-3). Subsequently, 40 g of the treated quartz sand was weighed out and used for test item analysis on study day -2. Thereafter, the remaining amount of the prepared quartz sand (240 g) was thoroughly mixed into the pre-moistened sediment (moisture 30 %) to ensure a homogenous distribution of the test item.

3. Artificial sediment: The artificial sediment had the following composition:
- 4 % peat, air dried and finely ground (AURICH-WIESMOOR-TORFVERTRIEB GMBH, D-26639 Wiesmoor)
- 20 % kaolin, kaolinite content preferably > 30 % (ZIEGLER & CO. GMBH, D-95621 Wunsiedel)
- 76 % quartz sand, sand with > 50 % particles sized 50-200 µm (DÖRENTRUP QUARZ GMBH, D-31089 Duingen)
- 0.35 % CaCO3, (ROTH)
The pH of the sediments was 6.66 in phase 1 and 3 and 6.67 in phase 2. Adjustment of the pH value was not necessary. The organic carbon content of the final mixture was measured on 2017-06-13 to be 2.24% (phase 1), 2.47% (phase 2) and 2.49% (phase 3) at AGROLAB Agrar und Umwelt GmbH, Sarstedt, Germany).

4. Test concentrations: The test item Disperse is not completely soluble in Tetrahydrofuran (THF) at the required concentration level for the maximum application concentration (1000 mg/kg dry weight) recommended by the guideline OECD 233. Therefore, a test design with a saturated solution in an extended limit test with 3 concentrations was used as a worst-case scenario.

Test organisms (species):

Chironomus riparius

Details on test organisms:

Test system Chironomus riparius (Arthropoda, Insecta, Pterygota, Diptera, Nematocera, Chironomidae)

Origin Own breeding, originally received from AQUATIC RESEARCH ORGANISMS INC, Hampton NH 03843, USA

Breeding Breeding was performed at the test facility at 20  2°C and diffuse light (500 - 1000 lx, 16 h photoperiod daily).
The dissolved oxygen concentration in the culture medium was ≥ 60% of the air saturation value (corresponding to 5.3 mg O2/L).
All midges used in the test originated from the same stock and were not used in another test.

Dilution water Medium M4, according to ELENDT (1990)

Feeding TetraMin® flake food (TETRA) was fed at least 3 times per week.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

59 d

Exposure phase:

total exposure duration

Remarks:

1st and 2nd generation

Hardness:

178 - 367 mg CaCO3/L

Test temperature:

19.3 - 22.2°C

pH:

7.7 - 8.5

Dissolved oxygen:

85 - 100% saturation

Salinity:

N/A

Ammonia:

0.17 - 22.4 mg/L

Conductivity:

N/A

Nominal and measured concentrations:

Nominal: 0 (control) - 25% - 50% - 100% of saturated solution applied to sediment (nominal 6.00 g/L stock solution)

Details on test conditions:

Replicates 10 replicates in total (8 for the biological part, 2 for the analytical part)

Control Control vessels without test item were included in the test with the same number of replicates.

Solvent control 10 replicates (8 for the biological part, 2 for the analytical part) with 20 larvae each (for the biological part and analytical part day 7) without the addition of the test item but treated with the solvent was tested under the same test conditions as the replicates with the test item.

Test method Three experimental phases were performed:
1) Insertion of larvae (1st instar) into test vessels until hatching of adult midges (1st generation)
2) Insertion of adult midges from phase 1 into breeding cages and obtaining larvae from egg ropes (1st to 2nd generation)
3) Insertion of larvae from phase 2 into test vessels until hatching of adult midges (2nd generation)

Test duration The maximum exposure duration for the 1st and 2nd generation was 28 days. The complete test duration was 59 days after the first test item application depending on the development of the test organisms.


Test vessel Glass beakers with a volume of 1 L and an inner diameter of 9 cm covered by gauze (phase 1: day 7, phase 3: day 0) after application were used. This size of the test vessel is sufficient to provide approximately 3 cm2 sediment surface per larvae.

Test sediment amount/height 180 g dry weight per vessel / approx. 2.0 cm from the ground.
height

Dilution water Same as for holding, amount of 550 mL: 550 mL by filling up the test vessels. The total hardness and the pH of the dilution water was measured 2 days before the start of the respective exposure phase.

Depth sediment-water The ratio of the depth of the sediment layer to the depth of the overlying water was ca. 1:4

Preparation of test vessels The treated sediment was filled into the test vessels. The dilution water was added to the vessels taking care not to disturb the sediment. Gentle aeration of the aqueous layer was provided through a glass Pasteur pipette. Water levels were topped to the original volume in order to compensate for water evaporation using deionised water. Equilibration times of 48 hours in phase 1 and 10 – 14 days in phase 3 were provided.
 

Collecting of egg masses/ Sufficient egg masses were collected and carefully
Introduction of the larvae/ transferred into glass vessels filled with dilution water.
Aeration Eggs were incubated until hatching. The larvae were inserted in 48 h old test vessels (phase 1) and in 10 – 14 days old test vessels (phase 3). The aeration of the test vessels was stopped during the introduction of the larvae and for the following 24 h. Gentle aeration of the overlying water in the test vessels was supplied 24 hours after addition of the first instar larvae and was continued throughout the test.

Age of the larvae First larval stage (2 days old)

Number of test larvae 180 first instar larvae were used per test concentration and control (20 larvae per replicate, 8 replicates each). The replicates for the analytical measurement on day 7 also received 20 larvae per vessel.

Feeding Food (TetraMin® flake food, TETRA) was provided once per per day. The amount of food was 0.25 - 1 mg per day and larvae, depending on the developmental stage.

Water temperature 20 ± 2°C

Dissolved oxygen Not less than 60% of the air saturation value
concentration

Light intensity 500 – 1000 lx

Photoperiod 16 h daily

Reference substance (positive control):

yes

Remarks:

potassium chloride (OECD 219, 28-d exposure)

Duration:

59 d

Dose descriptor:

NOEC

Effect conc.:

25 other: saturation % of saturated solution (6.00 g/L in THF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: 1st generation

Key result

Duration:

59 d

Dose descriptor:

EC10

Effect conc.:

30.9 other: saturation % of saturated solution (6.00 g/L in THF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Remarks on result:

other: 1st generation

Remarks:

95%-CI: 16.8-41.2%

Key result

Duration:

59 d

Dose descriptor:

NOEC

Effect conc.:

50 other: saturation % of saturated solution (6.00 g/L in THF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks on result:

other: 1st generation

Key result

Duration:

59 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: saturation % of saturated solution (6.00 g/L in THF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

sex ratio

Remarks on result:

other: 1st and 2nd generation

Key result

Duration:

59 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: saturation % of saturated solution (6.00 g/L in THF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

fecundity

Remarks on result:

other: 1st and 2nd generation

Key result

Duration:

59 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: saturation % of saturated solution (6.00 g/L in THF)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

fertility

Remarks on result:

other: 1st and 2nd generation

Details on results:

see table below

Results with reference substance (positive control):

- Results with reference substance valid: yes
- Relevant effect levels: 28-d EC50 (mortality): 3.70 g/L (95%-CI: 2.74 - 4.82 g/L)
- Dose-response test: yes
- Valid range: 3.40 +/- 1.85 g KCl/L

The test item was found to have an impact on the emergence rate (1st and 2nd generation) and the development rate (1st generation) in a long-time exposure on the non-biting midge Chironomus riparius. Effect values are given in the following table:

 

Effect levels based on saturation % of saturated solution

	1^stGeneration (Both Sexes)					2^ndGeneration (Both Sexes)
	Emergence Rate	Develop-ment Rate	Fecundity	Fertility	Sex Ratio	Emergence Rate	Develop-ment Rate	Sex Ratio
NOEC	25	50	100	100	100	50	100	100
LOEC	50	100	≥100	≥100	≥100	100	≥100	≥100
Ec10(95%CI)	30.9 (16.8 – 41.2)	>100	>100	>100	>100	>100	>100	>100
EC20(95%CI)	65.5 (51.7 – 83.0)	>100	>100	>100	>100	>100	>100	>100
EC50	>100	>100	>100	>100	>100	>100	>100	>100

Whereas statistical significant effects were found in the test concentrations compared to control in the 1^stGeneration, EC-values could only be calculated for the emergence rate. In the 2^ndGeneration, statistically significant differences compared to control were only found for the emergence rate at 100% saturation.

Therefore, the test item is considered to have only a minor effect on development and reproduction on Chironomus riparius up to the maximum achievable concentration in sediment. The emergence rate seems to be affected specifically, with varying severity at the same concentration However, no effect on the reproductive success was found from the 1^stto the 2^ndgeneration. The substance is not expected to cause long-term effects on the population stability of Chironomus riparius.

The stock solutions, Quartz sand samples, the control and the solvent control were analytically verified via LC-MS/MS on day -2. The samples of the water layer, the pore water and the sediment of the test item were analytically verified via LC-MS/MS during Phase 1 to 3 in all concentrations, the control and the solvent control. Hardly any substance could be found in the overlying and pore water. The substance remains in the spiked sediment. Analytical results and details of the method are attached in the study report.

Validity criteria fulfilled:

yes

Conclusions:

The biological results indicated effects on the emergence and development rate of C. riparius. In the 1st generation, NOEC/LOEC and ECx values could be determined for the emergence rate. A NOEC could be determined for the development rate for both sexes. No biologically significant effects were observed for the other endpoints fertility, fecundity and sex ratio. In the 2nd generation, only a NOEC on the emergence rate could be determined; other effects seen in the 1st generation were not replicable in the 2nd. The solvent THF did not cause any effects compared to the negative control. The NOECs of all other endpoints were >100% saturation. The biologically relevant and dose-related effect on the emergence rate in the 1st generation was determined with a LOEC of 50% of the saturated solution applied to the sediment. The EC10 was calculated at 30.9% (corresponding to nominal 1.854 g/L in the stock solution) whereas an EC50 could not be determined up to the highest concentration level. An effect on the emergence rate could still be seen in the 2nd generation but only at the highest concentration level. The NOEC for the emergence rate in the 2nd generation was found at 50% of the saturated solution. A slightly lower development rate in the highest test concentration led to a NOEC of 50% of the saturated solution in the 1st generation. This effect was no longer visible in the 2nd generation.

Executive summary:

The test item is considered to have only a minor effect on development and reproduction on C. riparius up to the maximum achievable concentration in sediment. Both emergence and development rate effects seem to attenuate from one generation to the next one. The emergence rate seems to be affected specifically, with varying severity at the same concentration. However, no effect on the reproductive success was found from the 1^stto the 2^ndgeneration. The substance is not expected to cause long-term effects on the population stability of C. riparius. The lowest relevant effect is the NOEC (1st generation) of the emergence rate at 25% saturation of the saturated stock solution with 6.00 g/L (corresponding to 1.854 g/L in the stock solution and to 0.417 mg/kg sed. as geometric mean measured concentration in the sediment).

Description of key information

The test item is considered to have only a minor effect on development and reproduction onC. ripariusup to the maximum achievable concentration in sediment. Both emergence and development rate effects seem to attenuate from one generation to the next one. The emergence rate seems to be affected specifically, with varying severity at the same concentration. However, no effect on the reproductive success was found from the 1^stto the 2^ndgeneration. The substance is not expected to cause long-term effects on the population stability ofC. riparius.The lowest relevant effect is: EC10 (1st generation) of the emergence rate at 30.9% saturation of the saturated stock solution with 6.00 g/L (corresponding to 1.854 g/L in the stock solution and to 0.417 mg/kg sed. as geometric mean measured concentration in the sediment)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

0.417 mg/kg sediment dw