Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 January 2020 to 31 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 126, Short Guidance on the Threshold Approach for Acute Fish Toxicity

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: June 07th - June 10th, 2016 & July 13th, 2016 / Date of signature: January 03rd, 2017

Specific details on test material used for the study:

- Density (certified): 0,921 kg/dm3
- Water solubility: 1.055 µg/L (20 °C, pH 7.164) (determined at Noack Laboratorien GmbH, 181120TL/CWE18483)
- Storage conditions: 18-25°C, dark, in the tightly closed original container

Analytical monitoring:

yes

Details on sampling:

The limit loading rate and the control was analytically verified via GC-MS/MS in the fresh media at the start of exposure and at one renewal interval (0 and 72 hours) and in the corresponding 24-hours aged media after 24 and 96 hours.
- Sampling method: Samples were taken directly from the test vessels.
- Sample storage conditions before analysis:

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fractions (WAF) with a nominal loading of 10000 mg/L were prepared 120 ± 1 hour prior to the start of the exposure as well as 120 ± 1 hour prior to each renewal of the test solutions in a glass flask (rinsed with cyclohexane and demineralized water) with a lateral outlet. This procedure is in accordance with the OECD guidance document No. 23 (2018) and ASTM D6081 (2014). For the limit loading rate, an appropriate amount of the test item (the density of the test item of 0.921 kg/dm3 was taken into account) was placed by pipette onto the surface of an appropriate amount of dilution water.
A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 120 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for 15 minutes at room temperature. Then, the WAF was removed by the lateral outlet of the glass flask from the approximate bottom of the water body. This procedure should not cause droplets by eye. The mixing regime was selected based on the observations during the acute daphnia toxicity test (Noack ID 181120TL/DAI18483). Three flasks of the WAF (nominal loading 10000 mg/L) were prepared for the day of start of the exposure and per renewal day.
- Differential loading: No. The WAF were tested in a limit test as a threshold loading. The loading is based on the derivation of a threshold loading (TL) from the results of an alga toxicity test and an acute daphnia toxicity test: no effects were observed in both studies, when tested with the WAF (highest nominal loading of 10000 mg/L).
- Controls: 7 fish in dilution water (without test item but treated in the same way as the test solution) were tested under the same test conditions as the test replicates.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The WAF was checked via laser beam (Tyndall effect) for undissolved test item and microscopically for dispersed droplets (phase-contrast microscope, magnification: 400 times). No presence of dispersed droplets or undissolved test item in the WAF was observed.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Danio rerio (zebrafish)
- Source: All fish used in the test were gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Weight at study initiation (mean and range, SD):
- Method of holding: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value. Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test. No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
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 = 40 - 250 mg CaCO3/L; pH-value = 6.0 - 8.5; Oxygen saturation ≥ 80 % of air saturation value; Acidity = 0.2 mmol/L (recent measurement 2020-01-21); Alkalinity = 0.6 mmol/L (recent measurement 2020-01-21); Conductivity = 149 µS/cm (recent measurement 2020-01-21)

ACCLIMATION
Acclimatization was not necessary, because the quality of the dilution water was equivalent to the water used for holding.

FEEDING DURING TEST
- Food type: Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany
- Feeding: Food was provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. The test fish were not fed 24 h before test start.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

62 mg CaCO3/L

Test temperature:

From 22.0 to 23.0°C

pH:

From 7.11 to 7.74

Dissolved oxygen:

97 to 99 % of air saturation value

Salinity:

Not applicable

Conductivity:

149 µS/cm (recent measurement 2020-01-21)

Nominal and measured concentrations:

- Nominal loading rate: 10000 mg/L
- Measured concentrations: See table 6.1.1/3 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with a glass plates
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Test volume about 2 L per vessel
- Aeration: Gentle aeration was provided.
- Renewal rate of test solution (frequency/flow rate): daily renewal of the test media
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same as used for holding

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A photoperiod (light / dark cycle: 16 / 8 h) was maintained during exposure.
- Light intensity: Light intensity was measured at the start of the exposure and ranged from 548 to 705 (mean value 628) Lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were made after 2 ± 0.5 h, 5 ± 1 h and 24 ± 2 h after the start of the exposure. On days 2-4 of the test, all vessels with living fish were inspected twice per day.
No mortality, no presence of visible abnormalities in regard to equilibrium, appearance, swimming behavior, ventilatory behaviour function was observed.

TEST CONCENTRATIONS
The test was carried out as a limit test with a threshold concentration (TC) based on an algae and a daphnia toxicity of the test item. In both tests no effects were observed when tested with a nominal loading rate of 10000 mg/L.

Reference substance (positive control):

no

Remarks:

No reference item is recommended for this test according to the guidelines.

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

>= 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The test solution was visibly clear throughout the period of exposure.
The environmental conditions (pH-value, temperature and oxygen-saturation) were determined to be within the acceptable limits.
The threshold concentration of the test item at the concentration level of 10000 mg/L was found to have no lethal or toxic effects on zebrafish.
The concentration of the test item was analytically verified via GC-MS/MS in fresh media at the start of the exposure (0 hours and 72 hours) and in the corresponding old media at the end of the exposure (24 hours and 96 hours) in the limit test item loading rate and the control. As the active ingredient of the test item shows 9 significant peaks, a group evaluation of three major groups took place: group I with peaks 1 to 3, group II with peaks 4 to 6 and group III with peaks 7 to 9. The grouping was based on the retention times of the signals.
All effect values given are based on the nominal test item loading since the test item is a UVCB substance and mixtures cannot be related to concentrations.

Results with reference substance (positive control):

Not applicable

Sublethal observations / clinical signs:

Table 6.1.1/1: Observations of sub lethal effects and normal behavior in the test vessels

(n=7, number of fish)

Limit loading rate (mg/L)	Effect*	Number of fish affected at observation time (hours)
		2	4	23	46	50.5	70	74.5	93	96
10000	(1)	7	7	7	7	7	7	7	7	7
Control	(1)	7	7	7	7	7	7	7	7	7

*) The numbers in brackets correspond to the following observations:

(1) = Normal behavior

Table 6.1.1/2: Cumulative mortality (%) in the test vessels

Limit loading rate (mg/L)	Cumulative mortality (%) at observation time (hours)
	2	4	23	46	50.5	70	74.5	93	96
10000	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0

Table 6.1.1/3: Measured concentrations of the test item during the definitive test

Sampling date	Fresh medium 0 hours				Old medium 24 hours
Nominal loading rate of the test item (mg/L)	Calculated concentration* (µg/L)				Calculated concentration* (µg/L)
	Group I	Group II	Group III	Sum**	Group I	Group II	Group III	Sum**
10000 Control QC	0.249 <LOQ RR: 89%	<LOQ <LOQ -	<LOQ <LOQ -	0.249 <LOQ -	0.128 <LOQ RR : 92%	<LOQ <LOQ -	<LOQ <LOQ -	0.128 <LOQ -
Sampling date	Fresh medium 48 hours				Old medium 72 hours
Nominal loading rate of the test item (mg/L)	Calculated concentration* (µg/L)				Calculated concentration* (µg/L)
	Group I	Group II	Group III	Sum**	Group I	Group II	Group III	Sum**
10000 Control QC	0.121 <LOQ RR: 112%	<LOQ <LOQ -	<LOQ <LOQ -	0.121 <LOQ -	0.0460 <LOQ RR: 84%	<LOQ <LOQ -	<LOQ <LOQ -	0.0460 <LOQ -

* Group I (Peak 1 to 3); group II (Peak 4 to 6); group III (Peak 7 to 9). Calculated concentration taking enrichment, dilution factor and the percentage of each group into account.

** Sum of group I to III, only measured values > LOQ taken into account

LOQ = Limit of quantification, group I: 0.1 µg test item/L, corresponding to 0.024 µg a.i./L; group II: 0.3 µg test item/L, corresponding to 0.14 µg a.i./L, group III: 0.6 µg/L test item/L, corresponding to 0.13 µg a.i./L

QC = quality control (0.1 µg test item/L), recovery calculated for group I to nominal concentration

- = not calculated

Validity criteria fulfilled:

yes

Conclusions:

The test item does not show toxic effects on fish at the limit loading rate of 10000 mg/L. Therefore, the 96h-LL50 value was determined to be > 10000 mg test item/L, based on nominal loading rates.

Executive summary:

The acute toxicity of the test substance to fish (zebrafish) was determined according to the principles of the OECD Guideline 203 (2019) from 2020 -01 -22 to 2020 -01 -31, with the definitive exposure phase from 2020 -01 -27 to 2020 -01 -31 at the test facility. 

Since the test item is a UVCB substance with compounds of different water solubility and due to the low aqueous solubility of the test item, the test item solution was prepared as a water accommodated fractions (WAF). The study was performed over a period of 96 hours under semi-static conditions with renewal of test solutions after every 24 hours as a worst-case exposure. The WAFs were clear. No dispersed droplets or undissolved test item was present in any of the tested WAFs. This was checked microscopically (phase-contrast microscope, magnification: 400 times). Seven test organisms were exposed to the test loading and the control, respectively. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The concentration of the test substance was analytically verified via GC-MS/MS in fresh media at the start of the exposure (0 hours and 72 hours) and in the corresponding old media at the end of the exposure (24 hours and 96 hours) in the limit test item loading rate and the control. As the active ingredient of the test item shows 9 significant peaks, a group evaluation of three major groups took place: group I with peaks 1 to 3, group II with peaks 4 to 6 and group III with peaks 7 to 9. The grouping was based on the retention times of the signals. 

All validity criteria of the test guideline were met. The test item does not show toxic effects on fish at the limit loading rate of 10000 mg/L.  

Description of key information

OECD Guideline 203, GLP, key study, validity 1:

96h-LL50 (Danio rerio) > 10000 mg/L based on nominal loading rates

Additional information

One key study is available to assess the short-term toxicity of the registered substance to fish.

In this acute toxicity test with Danio rerio (NOACK, 2020), the effects of one water accommodated fraction (WAF) with a nominal loading rate of 10000 mg/L of the registered substance were determined at the test facility according to OECD Guideline 203 with GLP compliance. A limit test with a threshold concentration was used based on the algae and acute daphnia toxicity tests. This study was conducted under semi-static and closed conditions over a period of 96 hours. No dispersed droplets or undissolved test item was present in the tested WAF. The concentration of the test substance was analytically verified via GC-MS/MS in fresh media at the start of the exposure (0 hours and 72 hours) and in the corresponding old media at the end of the exposure (24 hours and 96 hours) in the limit test item loading rate and the control. According to the results of this study, the test substance does not show toxic effects on fish at the limit loading rate of 10000 mg/L. Therefore, the 96h-LL50 value was determined to be > 10000 mg test item/L, based on nominal loading rate.