Registration Dossier

Ecotoxicological information

Sediment toxicity

Currently viewing:

Administrative data

Endpoint:
sediment toxicity: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 09 November 2016 Experimental completion date 20 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Alkyl (C12-C14) glycidyl ether
Label: Epoxide 8
51% C12 Glycidyl ether
21% C14 Glycidyl ether
5% C16 Glycidyl ether
13% N +1's
4% alpha addition Products
94% total active (label claim)
- Physical state: Clear colorless liquid
- Storage condition of test material: ambient
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification:
Oxirane, mono[(C12-14-alkyloxy)methyl] derivs
CAS-No:
68609-97-2
Batch Number:
AAD1326100
Physical state/Appearance:
Clear colorless liquid
Molecular Formula:
C4H10O.x(C3H6O.C2H4O)x.xl2
Purity:
UVCB
Expiry Date:
29 August 2019
Storage Conditions:
Stored cold at ≈ 4 ºC in the dark

Sampling and analysis

Details on sampling:
SEDIMENT
A defined formulated sediment was used with the following composition:
Industrial quartz sand 76% w/w
Kaolinite clay 20% w/w
Sphagnum moss peat 4% w/w
In the definitive test, for the control, solvent control and each test concentration, a peat suspension was prepared by homogenising 60 g of peat and 450 mL of deionized water in a high shear mixer at 1300 rpm for 10 minutes. The pH of the suspension was adjusted using calcium carbonate to 5.5 ± 0.5 and was placed set to stir using a magnetic stirrer for approximately 2 days. At the end of the stirring period, the pH was measured and adjusted using calcium carbonate to 6.0 ± 0.5. The remaining dry constituents (prepared as seven batches, each of 1140 g sand and 300 g clay) were combined with the peat suspension, plus an additional 150 mL of deionized water and thoroughly mixed using a large scale laboratory mixer. The pH of each batch of the final sediment was measured and adjusted to 7.0 ± 0.5.
Each batch of sediment was transferred to a 5 liter glass beaker, forming a sediment layer of approximately 5.5 cm. Elendt M4 media was carefully added forming an overlying water layer approximately 12 cm deep. The sediment/media preparations were covered and aeration was initiated at a rate of approximately 1 to 2 bubbles per second. The sediment preparations were allowed to condition under test conditions for 11 days prior to use in the study. At the end of the conditioning period, the overlying water was decanted and disposed of, the test item mixed with the sediment and fresh media was added as overlying water.

WATER
The reconstituted water (Elendt M4) used for the range-finding and definitive tests was the same as that used to maintain the stock animals.
Dissolved oxygen concentrations, water temperature and pH were recorded weekly in each test vessel throughout the test. The water hardness and ammonia concentration was determined in one vessel from the solvent control and 1000 mg/kg on Day 0 and in one vessel from the control, solvent control and 1000 mg/kg on Day 28 using the methods described in Fields and On-Site Methods for Analysis of Water (British Standards Institution, 1993).

Test substrate

Vehicle:
no

Test organisms

Test organisms (species):
Chironomus riparius
Details on test organisms:
The test was carried out using larvae (2 to 3 days old) of Chironomus riparius derived from in-house laboratory cultures.
Larvae were maintained in glass beakers with a 5-10 mm layer of fine quartz sand covered by reconstituted water (Elendt M4) in a temperature controlled room at approximately 20 ºC. The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods. The cultures were gently aerated, so as not to disturb the substrate, through narrow bore glass tubes. The culture vessels were housed in a sealed clear perspex cabinet (breeding box) with cotton sleeves to enable access.
The larvae were fed with Tetramin® flake food at approximately 250 mg per vessel per day. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.
Any gelatinous egg masses produced by breeding adult midges were removed from the culture vessels and transferred to separate vessels and, if required, larvae used to populate new cultures at an initial density 200 larvae.
The diet and diluent were considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Study type:
laboratory study
Test type:
static
Water media type:
other: reconstituted water (Elendt M4)
Type of sediment:
artificial sediment

Test conditions

Hardness:
The water hardness values was determined to be 280 mg/L as CaCO3 for the control on Day 28, 248 and 260 mg/L as CaCO3 for the solvent control on Days 0 and 28 respectively, and 268 and 356 mg/L as CaCO3 for the 792 mg/kg test group on Days 0 and 28 respectively. The theoretical hardness of the Elendt M4 medium is 250 mg/L as CaCO3. The increase in hardness was considered to be due to the addition of calcium carbonate to the sediment during preparation to adjust the pH to 7.0 ± 0.5.
Test temperature:
Temperature, as recorded by the Testo data logger, was maintained at 20.0 °C to 22.1 °C throughout the test
pH:
The pH values in the water columns should be between pH 6 and 9 in all test vessels.
Ammonia:
The ammonia concentrations were determined to be 0.0019 and 0.00047 mg/L as NH3 for the solvent control on Days 0 and 28 respectively and 0.0017 and 0.00041 mg/L as NH3 for the 792 mg/kg test group on Days 0 and 28 respectively.
Details on test conditions:
3.4.2 Initial Test
Based on the results of the range-finding test an initial test was conducted as a "limit test" at a concentration of 1000 mg/kg (dry weight of sediment), however, results of the limit test showed a significant effect in terms of the development rate therefore a full range definitive test was conducted.

3.4.3 Definitive Test
Based on the results of the initial test the following concentrations were assigned to the definitive test: 62.5, 125, 250, 500 and 1000 mg/kg (dry weight of sediment).
3.4.3.1 Experimental Preparation
Approximately two days prior to the start of the test, a nominal amount of test item (3750 mg) was dissolved in acetone and the volume adjusted to 25 mL to give a 150 mg/mL stock solution. A series of dilutions was performed to give further stock solutions of 75, 37.5, 18.75 and 9.375 mg/mL in acetone.
Aliquots (10 mL) of each stock solution were each separately added to 20 g of sand and the acetone evaporated off at room temperature. After the acetone had been evaporated off the spiked sand was mixed with 1500 g (dry weight) of formulated sediment and mixed with a Kenwood chef mixer to give the test concentrations of 62.5, 125, 250, 500 and 1000 mg/kg. The solvent control and control were prepared in a similar manner, without the addition of test item or solvent for the control and without the addition of test item for the solvent control.
The prepared sediment was dispensed to glass beakers to give a 2 cm layer and was then covered with an 8 cm depth of test water (sediment:water ratio, 1:4).
Four replicates vessels were prepared for the control, solvent control and each test concentration for incubation over the 28-Day exposure period. A plastic disc was placed over the sediment and the test water poured gently onto the surface of the disc in order to avoid disturbance of the sediment. The disc was removed after addition of the water and the vessels allowed to settle for 1 day. After the settlement period, the test vessels were then aerated (approximately 1 bubble/second) via narrow bore glass tubes approximately 2 to 3 cm above the sediment layer and the vessels left for 1 day prior to addition of the test organisms in order to allow settlement and equilibration of test concentrations between the sediment and water phases.
An additional two replicates were prepared for the solvent control, 62.5 and 1000 mg/kg test concentrations to provide samples for chemical analysis on Days 0 and 7 and one further replicate was prepared for the control, solvent control and each test concentration to provide samples for chemical analysis on Day 28.
Analysis for the concentration of the test item in the sediment was also performed on Day -2 (the day of sediment preparation) to confirm correct dosing of the test system.
3.4.3.2 Exposure Conditions
As in the range-finding test 600 mL glass beakers approximately 8 cm in diameter were used. After the 2-Day equilibration period the aeration was stopped and 20 larvae were placed in each test and control vessel and maintained in a temperature controlled room at 20 ± 2 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk
transition periods. The aeration was switched back on after approximately 24 hours having allowed the larvae to settle in the sediment.
During the equilibration period 5 mg of Tetramin® flake food was added to each vessel to allow ingestion of contaminated food from the start of the test. The larvae were fed at a rate of 0.50 mg Tetramin® flake food per larva per day for the first 10 days and 1.0 mg Tetramin® flake food per larva per day thereafter. The Tetramin® flake food was prepared as a suspension in water and an appropriate volume added to the overlying water.
The control group was maintained under identical conditions but not exposed to the test item.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 792 mg/kg sediment dw
Conc. based on:
test mat.
Basis for effect:
emergence rate
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 792 mg/kg sediment dw
Conc. based on:
test mat.
Basis for effect:
development time
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
105 mg/kg sediment dw
Conc. based on:
test mat.
Basis for effect:
emergence rate
Details on results:
Measured test concentrations of less than the Limit of Quantification (LOQ) of the analytical method were determined for a number of samples. The LOQ was determined to be 0.058 mg/L for overlying water, 0.58 mg/L for interstitial water and 5.84 mg/kg for test sediment. Measured concentrations reported as less than the LOQ do not infer that no test item was present in the sample, just that any test item was at a concentration of less than the LOQ.
The measured concentrations in the sediment were not within ±20% of the nominal concentrations. It is believed that the low measured concentrations were as a result of the test item binding to the sediment, therefore, Day -2 measured concentrations have been used in the evaluation of study end-points.
Analysis of sediment, overlying water and pore water samples indicated that the test item mostly remained in the sediment phase throughout the duration of the test.

Examination of the numbers of male and female adults midges emerged (see Tables 3 to 9) showed no biologically significant difference between the numbers of male and females. The sex ratio of the emerged adult midges was statistically analyzed (see Appendix 1) and showed there was no statistically significant difference between the number of males and females emerging. Therefore, it was considered that the test item had no significant effect on the sex ratio of emerged adults.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test item to the sediment-dwelling larvae of Chironomus riparius has been investigated.
Measured concentrations of the test item in the sediment two days before the addition of the larvae were 50, 105, 189, 358 and 792 mg/kg respectively, and these values were used in the evaluation of study end-points. Measured concentrations of test substance in the sediment at Day 28 gave values of 6.4, 11, 27, 66 and 304 mg/kg, demonstrating that concentrations had not been maintained throughout the test.
Analysis of sediment, overlying water and pore water samples indicated that the test item mostly remained in the sediment phase throughout the duration of the test.
Executive summary:

A study was performed to assess the toxicity of the test item to the sediment-dwelling larvae of Chironomus riparius, using a static test system with application of radiolabelled test substance to the sediment phase.

The method followed recommendations of the OECD Guidelines for Testing of Chemicals No. 218 “Sediment-Water Chironomid Toxicity Test using Spiked Sediment” (Adopted April 2004).

1.2 Methods

Following a preliminary range-finding test larvae of Chironomus riparius were exposed in groups of 80 (four replicates of 20 larvae per concentration) to formulated sediment spiked with test item over a range of concentrations of 62.5, 125, 250, 500 and 1000 mg/kg for a period of 28 days. The numbers of emerged adult midges were recorded daily.

1.3 Results

Measured concentrations in the sediment two days before the addition of the larvae were 50, 105, 189, 358 and 792 mg/kg respectively, and these values were used in the evaluation of study end-points. Measured concentrations of test substance in the sediment at Day 28 gave values of 6.4, 11, 27, 66 and 304 mg/kg, demonstrating that concentrations had not been maintained throughout the test.

A summary of the study end-points is given below:

 End Point  EC10 (mg/kg)  EC20 (mg/kg)  EC50(mg/kg)  NOEC (mg/kg) LOEC (mg/kg)
 Reduction in emergence 191   604  >792  105  189
 Development rate-males  >792  >792  >792  792  >792
 Development rate-females  >792  >792  >792  792  >792
 Development rate-combined  >792  >792  >792  792  >792

Analysis of sediment, overlying water and pore water samples indicated that the test item mostly remained in the sediment phase throughout the duration of the test, however, measured concentrations in the sediment were observed to decline throughout the duration of the test.