Fatty acids, C16-18, reaction products with diethanolamine

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:

2013-03-01 to 2013-03-08

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A water accommodated fraction (WAF) of 100 mg/L was prepared with dilution water 24 h prior to application.
- Sampling method: Analytical evaluation of the limit loading level of Fatty acids, C16-18-, reaction products with diethanolamine and the control was carried out via LC-MS/MS at start and end of the exposure
- Sample storage conditions before analysis:All original samples were stored room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A water accommodated fraction (WAF) of 100 mg/L was prepared with dilution water 24 h prior to application. Weighing of the test item for preparation was carried out 3 days prior to the start of the exposure. The weighted amount was stored in the dark and at room temperature until preparation of the WAF.
The test item dispersion was shaken for 24 hours at 20 rpm (rotating shaker 3040, GFL). After the separation of the phases, after 1.5 hour of standing, the WAF was drawn off from the homogenous liquid phase (middle of the test item solution) and used for testing without further dilution.
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicate.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test originate from a single brood stock at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Niedersächsischer Landesbetrieb für Wasserwirtschaft, Küsten- und Naturschutz, An der Scharlake 39, D-31135 Hildesheim)
- Length at study initiation (length definition, mean, range and SD): Average body length: 2.7 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.21 g
- Method of breeding: Holding was performed at the test facility at 23+/-2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test



ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 45 mg/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open, loosely covered by glass tops
- Material, size, headspace, fill volume: glass, 10 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): application at test start
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: less than 1 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Same as for holding
- Culture medium different from test medium: 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
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at the beginning out of the test control. During the test the water temperature was recorded continuously with a data logger (once per hour).


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: natural
- Light intensity: 0.1 - 10 µmol photons x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study:

Cumulative Mortality [%] in the Preliminary Test
(n = 5)
Nominal test item
loading level
[mg/L] Test Duration [hours]
24 48 72 96
100 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item was clearly dissolved in the tested loading levels during exposure.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

The test loading leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test. LC50-values have not to be calculated in a limit test.

Sublethal observations / clinical signs:

Observations in the Test Vessels

Nominal test item loading level [mg/L]	Effect*	Test Duration [h]
		2	24	48	72	96
100	(1)	7/7	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7	7/7

Cumulative Mortality [%] in the Test Vessels

Nominal test item loading level [mg/L]	Test Duration [h]
	2	24	48	72	96
100	0	0	0	0	0
Control	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

In this study Fatty acids, C16-18-, reaction products with diethanolamine was found to cause no toxic effects to Zebrafish after 96 hours when tested with the water accommodated fraction with a nominal loading rate of 100 mg/L.
The LL50 after 96 hours is > 100 mg/L All effect levels are based on the nominal loading level of the test item.

Executive summary:

The acute toxicity of the test item Fatty acids, C16-18-, reaction products with diethanolamine to fish (zebrafish), was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992)  from 2013-03-01 to 2013-03-08, with the definitive exposure phase from 2013-03-04 to 2013-03-08 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A static test with the water accommodated fraction (WAF)* with a nominal loading of 100 mg/L was performed. The duration of the test was 96 hours. 7 test organisms were exposed per limit loading and control, respectively. Water quality parameters temperature, pH-value and O₂-saturation measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The test item Fatty acids, C16-18-, reaction products with diethanolaminewas analytically verified by LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (96 h) of the WAF with a nominal loading of 100 mg/L and the control.

The measured concentration of the test item in the WAF at the start of the exposure (0 h) was 0.725 mg/L. The measured concentration in the WAF at the end of the exposure (96 h) was 0.149 mg/L. The analytical results are presented in Table 8. All effect levels are given based on the nominal loading level of the test item (see Table 1).

Table 1:    LL-Values with 95 % Confidence Intervals (0 – 96 hours)

                   Based on the nominal test item loading [mg/L]

Test duration [h]	Nominal test item loading [mg/L]
	LL50	p = 95 %
24	> 100	n.a.
48	> 100	n.a.
72	> 100	n.a.
96	> 100	n.a.
LL100 = Lowest test item loading with 100 % mortality after 96 h	> 100
LL0 = Highest test item loading with 0 % mortality after 96 h	100

Description of key information

In the key study Fatty acids, C16-18-, reaction products with diethanolamine was found to cause no toxic effects to Zebrafish after 96 hours when tested with the water accommodated fraction with a nominal loading rate of 100 mg/L.

The LL50 after 96 hours is > 100 mg/L All effect levels are based on the nominal loading level of the test item.

In a supporting study the LC50of the test item is estimated to be greater than the applied nominal test concentration of 100 mg/|.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

KEY study

The acute toxicity of the test item Fattyacids,C16-18-, reactionproducts with diethanolamine to fish (zebrafish), was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2013-03-01 to 2013-03-08, with the definitive exposure phase from 2013-03-04 to 2013-03-08atDr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A static test with the water accommodated fraction (WAF)* with a nominal loading of 100 mg/L was performed. The duration of the test was 96 hours. 7 test organisms were exposed per limit loading and control, respectively. Water quality parameters temperature, pH-value and O₂-saturation measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The test itemFattyacids, C16-18-, reactionproducts with diethanolaminewas analytically verified by LC-MS/MSat the start of the exposure(0 h) and at the end of the exposure (96 h) of the WAF with a nominal loading of 100 mg/L and the control. Details for the analytical method are presented in part 8.

The measured concentration of the test item in the WAF at the start of the exposure (0 h) was 0.725 mg/L. The measured concentration in the WAF at the end of the exposure (96 h) was 0.149 mg/L. The analytical results are presented inTable8.All effect levels are given based on the nominal loading level of the test item (seeTable1).

 

Table1:   LL-Valueswith 95 % Confidence Intervals (0 – 96 hours)

                  Based on the nominal test item loading [mg/L]

 

Test duration [h]	Nominal test item loading [mg/L]
	LL50	p = 95 %
24	> 100	n.a.
48	> 100	n.a.
72	> 100	n.a.
96	> 100	n.a.
LL100= Lowest test item loading with 100 % mortality after 96 h	> 100
LL0= Highest test item loading with 0 % mortality after 96 h	100

 

In a supporting study the LC50of the test item is estimated to be greater than the applied nominal test concentration of 100 mg/L.