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Reference
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-06 to 2017-08-04
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 Disperse Yellow DYLA 1306 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 Disperse Yellow DYLA 1306 were analytically verified after an equilibration time of at least 48 hours 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 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 – 35 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 1.5 - 1.75 hours. 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 6.8 in all phases. Adjustment of the pH value was not necessary. The organic carbon content of the final mixture was measured on 2017-05-08 to be 2.68% (phase 1), 2.66% (phase 2) and 2.53% (phase 3). A mixed sediment from phase 1 and 2 was measured again on 2017-05-15 to be 2.35% at AGROLAB Agrar und Umwelt GmbH, Sarstedt, Germany).

4. Test concentrations: The test item Disperse Yellow DYLA 1306 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
Hardness:
297 - 351 mg CaCO3/L
Test temperature:
18.8 - 21.6°C
pH:
8.0 - 8.7
Dissolved oxygen:
85 - 100% saturation
Salinity:
N/A
Ammonia:
0.45 - 12.9 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)
Key result
Duration:
59 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: % of saturated solution (6.00 g/L in THF)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
emergence rate
Key result
Duration:
59 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: % of saturated solution (6.00 g/L in THF)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
development rate
Key result
Duration:
59 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 other: % of saturated solution (6.00 g/L in THF)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
sex ratio
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 Disperse Yellow DYLA 1306 was found to have an impact on the emergence 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

 

1stGeneration

2ndGeneration

 

Emergence

Develop-ment Rate

Fecundity

Fertility

Sex Ratio

Emergence

Develop-ment Rate

Sex Ratio

NOEC

25

≥100

≥100

≥100

≥100

≥100

≥100

≥100

LOEC

50

>100

>100

>100

>100

>100

>100

>100

 

 Only Emergence in the 1stgeneration showed a significant difference compared to control to a concentration of 50% and 100% saturated solution.

Emergence in the 2ndgeneration was not statistically significant different compared to control.

Furthermore, statistical significant effects were found to be non-dose-related (effects in 50% saturated solution but not 100% saturated solution) and not severe enough to calculate EC-values.

The emergence ratio and development rate in the 1stgeneration and emergence ratio and sex ratio in the 2ndgeneration were found to be statistically significant different compared to the controls in the test item replicates. Since the values for these endpoints are still inside the validity criteria for the control and seem to be non-dose-related, these deviations are considered as a biological effect not directly related to the test item.

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 Disperse Yellow DYLA 1306 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 test item Disperse Yellow 1306 did not cause any reproducible negative effects on emergence rate, development rate or sex ratio of Chironomus riparius. The substance is chronically not toxic to sediment dwelling organisms. No effects were seen in generation 1 in development rate, fecundity, fertility and sex ratio. A slight effects was found in the emergence rate (NOEC = 25% of the saturated solution). However, this effects was not found in generation 2 anymore.

Description of key information

The test item Disperse Yellow 1306 did not cause any reproducible negative effects on emergence rate, development rate or sex ratio of Chironomus riparius. The substance is chronically not toxic to sediment dwelling organisms. It was tested in a saturated solution (6.00 g/L in tetrahydrofuran) at 100%, 50% and 25% in spiked sediment.

Key value for chemical safety assessment

Additional information