Fatty acids, C18 (even numbered) and C18 unsatd., reaction products with triethanolamine, di-Me sulfate quaternized

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar (eco)toxicological properties because
• they share structural similarities with common functional groups: One quaternised ethanolamine moiety, one to three, mainly two ester groups with a typical UVCB distribution with long-chain fatty acids of natural origin. The molecular structure is almost identical.
• they are manufactured from similar resp. identical precursors (triethanolamine, long-chain fatty acids, dimethyl sulphate) under similar conditions. Therefore common breakdown products via physical and biological processes, which result in structurally similar chemicals are evident
• A constant pattern in the changing of the potency of the properties across the TEA-Esterquats by chain-length and the grade of esterification is not observed, because the fatty acid chain-length distribution is too narrow and similar and the distribution of mono-, di-, and tri-esters is identical. Some variation caused by variation in C=C double bonds may occur and will be discussed at the relevant endpoint.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See justification for read-across attached to chapter 13 of this IUCLID file.

3. ANALOGUE APPROACH JUSTIFICATION
See justification for read-across attached to chapter 13 of this IUCLID file.

4. DATA MATRIX
See justification for read-across attached to chapter 13 of this IUCLID file.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: All fish used in the test were reared at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin, Germany)

Maintenance of brood fish
A breeding stock of unexposed, mature zebrafish with an age of approx. 10 - 12 months were used for the egg production. Fish were free of macroscopically discernable symptoms of infection and disease. Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60% of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (7 – 750 lux on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum; Daphnia magna, juvenile and adult daphnids, ad libitum; dry food sera vipan SERA, ad libitum.
- No disease treatments were administered. 

Water Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 – 250 mg CaCO3/L
pH-value: 6.0 – 8.5
TOC: < 2 mgC/L
Alkalinity: 0.6 mmol/L (recent measurement: 2021-01-14)
Acidity: 0.2 mmol/L (recent measurement: 2021-01-14)
Conductivity: 176 µS/cm (recent measurement: 2021-01-14)

Spawning
15 – 35 adult zebrafish were kept in 3 separate aquaria. The fish were healthy with a mortality rate < 5% during the last 7 days and thus not medically treated for at least 7 days. About 15 minutes before start of artificial dawning rectangular dishes covered with a stainless-steel mesh and provided with artificial plants (plastic), were introduced into the aquaria. After 1.5 hours the glass dishes were gently removed. Eggs were checked carefully for abnormalities like fungus infections. These eggs as well as coagulated and not fertilized eggs were discarded (less than 30%). About 800 eggs were taken and washed in dilution water. Eggs originated from 3 different spawnings.

Start of exposure
The eggs that were used to start the exposure were pooled and attributed randomly (eggs were placed in alternating groups into each of the test groups) to the test groups in crystallization dishes containing test solutions (two dishes per test group, each dish loaded with at least 60 eggs, resulting in a total of minimum 120 eggs per test item loading rate).

Fertilization check
Immediately after exposing the eggs to the test solutions (start of exposure), the eggs were checked for fertilization. Eggs were fully covered with the respective test solutions. Every embryo was checked under a stereo microscope for its stage. Cleavages which form 4, 8, 16 and 32 cell blastomers could be clearly identified by the development of the blastula and were regarded to be fertilized. Eggs with only a 2 cell stage were regarded as not fertilized and discarded.

Fertilization rate
The mean fertilization rate was 95%.

Introduction of eggs
The eggs were placed onto gauze cages positioned in the middle of the water phase of the test vessels directly after the fertilization check at a stage before cleavage of the blastodisc commences or as close as possible to this stage. The eggs were transferred randomly into test vessels containing the respective exposure solutions. The distribution of eggs to the test item loading rates was carried out indiscriminately by adding 5 eggs to the first test group, the 2nd 5 eggs to the next test group and so on, until all test groups contained the necessary number of eggs.

Feeding of test fish
The feeding regime was ad libitum during the whole feeding period (study day 5 to 34).
Feeding started 2 days after the beginning of hatch on study day 5 (post-hatch day 1, where almost all non affected larvae swum up). Larvae were fed with a starter food (ST-1 (AQUA SCHWARZ GMBH, 37081 Göttingen, Germany), as well as a suspension of the starter food ST-1 and fine milled brine shrimp nauplii (2 – 7 times daily). 1 day after start of feeding brine shrimp nauplii (48 h old) were fed until the end of the test (2 – 7 times daily).
Brine shrimp nauplii origin, breeding conditions:
Artemia salina (Brine shrimp eggs) were purchased from Kessler Zoologiegroßhandel GmbH & Co. KG, D 67122 Altrip, Germany. Fresh cultures were prepared with salt water (NaCl 20 g/L, ca. 2 g eggs to 1 L salt water, gentle aeration for 24 - 48 hours at approx. 22 °C). 24 - 48 h old brine shrimp nauplii were harvested, washed in a stainless-steel mesh and resuspended in tap water. Feeding ad libitum was carried out

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Remarks on exposure duration:

30 days post hatch

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

0.582 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

Weight

Remarks on result:

other: 95% c.i. 0.287 – 1.18 mg/L

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

0.66 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

length

Remarks on result:

other: 95% c.i. 0.411 – 1.06 mg/L

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

1.79 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Hatching success after 6 days

Remarks on result:

other: 95% c.i. 0.581 - > 4.68 mg/L

Key result

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.224 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival and overall survival

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

0.461 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: fry growth (expressed as length and fresh weight)

Duration:

6 d

Dose descriptor:

NOEC

Effect conc.:

0.461 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: hatching success after 6 d

Description of key information

NOEC (Post-hatch survival, overall survival) = 0.224 (OECD TG 210, Danio rerio); GLP; RL1 (weighted arithmetic mean measured concentrations); read-across from partially unsaturated TEA-Esterquat

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.224 mg/L

Additional information

No experimental data on the long-term toxicity to fish are available for the target substance C18 and C18 unsatd. TEA-Esterquat. However, reliable relevant data are available for the closely related source substance partially unsaturated TEA-Esterquat.

A justification for read-across is attached to Iuclid section 13.

 

The effects of the test item partially unsatd TEA-Esterquat on the early-life stage of fish (Danio rerio/ Zebrafish) were determined according to OECD Guideline 210.

 

A test item dispersion with a dry content of 10.3% was prepared from the original test item. Stock dispersions in demineralized water with nominal concentrations of 8.00, 16.0, 32.0, 64.0 and 128 g dispersion/L, corresponding to 0.824, 1.65, 3.30, 6.59 and 13.2 g test item/L were prepared in appropriate intervals of 3 to 4 days and continuously dosed to the dilution water in a flow-through system. Based on the results of a range finding test the test was conducted as a dose-response test with the nominal test item loading rates of 4.00, 8.00, 16.0, 32.0 and 64.0 mg dispersion/L, corresponding to 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L ), corresponding to time weighted arithmetic mean measured concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L..

The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio/ zebrafish were exposed to each test item loading rate and the control (4 replicates with 20 eggs each).

The water quality parameters pH-value, oxygen concentration, temperature and total hardness were within the acceptable limits.

On study day 4, 95% of the control larvae had hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).

Different toxicological endpoints were determined: hatching success, fry growth (assessed via length and fresh weight measurements on PHD 30), morphological and behavioral effects, post-hatch survival and overall survival.

Specific analysis of various concentrations ofthe test item in the test media and the controls was carried out via LC-MS/MS.

The test media were sampled and analyzed from alternating test vessels prior to exposure on day -1 and during the exposure on study days 0, 7, 14, 21 and 28.

The measured concentrations of the test media during equilibration days -3 to -1 ranged between 70 and 109% of the nominal loading rates. The measured concentrations of the test media on study days 0 to 28 were in the range of 31 to 88% of the nominal loading rates.

The stock dispersions were sampled and analyzed from freshly prepared and corresponding 4 days aged stock dispersions. Measured concentrations of the freshly prepared stock dispersions were 92 to 99% of the nominal values. Measured concentrations of the 4 days aged stock dispersions in the range of 91 to 99% of the nominal values.

 

At the end of the exposure on study day 34 the aqueous phase of replicates used for analysis of glassware adsorption were analysed too. The sorption of the test item on glass was quantified at the loading rates of 4.00 and 16.0 mg dispersion/L from one replicate per test group at the end of the exposure and from one replicate the loading rate of 64 mg dispersion/L after 100 % mortality occurred.

 

Findings and Observations

The results of the parameters hatching success, fry growth (expressed as weight and length measurement at PHD 30), post-hatch survival and overall survival were checked for statistically significant differences.

The test item caused significant effects on Zebrafish in an early life stage test, 30 days post hatch when tested with nominal loading rates of 0.412, 0.824, 1.65, 3.30 and 6.59 mg test item/L, corresponding to the time weighted arithmetic mean measured (TWA) concentrations of 0.224, 0.461, 0.982, 2.60 and 4.68 mg test item/L.

For the parameter hatch, the NOEL was 0.824 mg test item/L (TWA: 0.461 mg test item/L). Therefore, the respective LOEL was determined to be 1.65 mg test item/L (TWA: 0.982 mg test item/L).

For the parameters post hatch survival and overall survival, the NOELs were 0.412 mg test item/L (TWA: 0.224 mg test item/L), respectively. Therefore, the respective LOELs were determined to be 0.824 mg test item/L (TWA: 0.461 mg test item/L).

For the parameter fry growth (expressed as length and fresh weight) the NOELs were 0.824 mg test item/L (TWA: 0.461 mg test item/L) for both parameters. Therefore, the LOELs for length and weight were determined to be 1.65 mg test item/L (TWA: 0.982 mg test item/L), respectively.