3-(isodecyloxy)propiononitrile

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-04 to 2013-03-08

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-(isodecyloxy)propiononitrile

EC Number:

264-840-1

EC Name:

3-(isodecyloxy)propiononitrile

Cas Number:

64354-92-3

Molecular formula:

C13H25NO

IUPAC Name:

3-(decylalkyl-(branched)oxy)-propiononitrile

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.00 - 1.77 - 3.17 - 5.64 - 10.0 mg/L (nominal) corresponding to geometric mean measured test item concentrations of 0.712 – 1.30 – 2.46 – 3.74 – 5.85 mg/L.

- Sampling method: Analytical evaluation of the various concentrations of 3-(Isodecyloxy)propionitrile was carried out via LC-MS/MS from freshly prepared media after 0 h and 72 h and from corresponding 24 h aged test media after 24 h and 96 h.
- Sample storage conditions before analysis: All original and diluted samples were stored at room temperature until sample preparation and until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prior weighing, the test item was treated with ultrasound for 5 minutes at 40 °C. Directly weighing. The test media was mixed with an ultraturrax (1 min, 19600 rpm).

- Eluate: Dilution water
- Differential loading: 1.00 - 1.77 - 3.17 - 5.64 - 10.0 mg/L (nominal) corresponding to geometric mean measured test item concentrations of 0.712 – 1.30 – 2.46 – 3.74 – 5.85 mg/L.
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms

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 were gained at DR.NOACK-LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

Total Hardness at day 0: 50 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): dimensions: 3 L glass aquaria, 11.5/13/20 cm, depth of water: 16.5 cm, the aquaria were covered with a floating lid of aluminium foil to reduce the loss of the test item.
- Material, size, headspace, fill volume: glass, 2 L test volume per vessel
- Aeration: No aeration
- Renewal rate of test solution (frequency/flow rate): daily renewal
- 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

Observations in the Preliminary Test
(n = 5)
Nominal test item
concentration
[mg/L] Effect *) Number of affected fish at observation time [hours]
24 48 72 96
10.0 (E) 5/5 - - -
5.00 (2.1) 5/5 2/5 2/5 2/5
(2.2) - 3/5 3/5 3/5
1.00 (1) 5/5 5/5 5/5 5/5
Control (1) 5/5 5/5 5/5 5/5
*) The number in brackets corresponds to the following observation:
(1) = Normal behaviour
(2.1) = Lethargy
(2.2) = Fish is lying on its side
(E) = Exitus letalis

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: (2.46 - 3-74)

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 solutions were clear throughout exposure.
In order to maintain the test item concentrations as far as possible, the definitive test was carried out under semi-static conditions with daily renewal of test media and without aeration. The test aquaria were covered with floating lids to avoid losses due to evaporation.

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

After 24, 72 and 96 hours only 0 and 100 % mortality occurred. Hence the LC50 after 24, 72, 96 hours is the geometric mean value of the geometric mean measured concentrations with 0 and 100 % mortality. The concentrations causing 0 and 100 % were chosen as lower and upper confidence limit, respectively. Therefore, LC10,20-values could only be given as a range of those concentrations.
After 48 hours 29 and 100 % mortality occurred. The LC10,20,50-values were calculated by non linear regression (sigmoidal dose response, variable slope). The chosen methods for the determination of LCx-values were in accordance with the OECD series on testing and assessment No. 54 (2006). The concentrations leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the raw data.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Observations in the Test Vessels

Geometrical mean measured concentration [mg/L]	Effect *	Number of fish effected at observation time [hours]
		24	48	72	96
5.85	(E)	7/7	---	---	---
3.74	(E)	-	2/7	5/5	---
	(4.1)	7/7	5/7	---	---
	(2.5)	3/7	-	---	---
	(2.2)	1/7	2/7	---	---
	(2.1)	3/7	3/7	---	---
2.46	(4.1)	7/7	7/7	7/7	7/7
	(2.5)	4/7	1/7	-	-
	(2.1)	3/7	6/7	7/7	7/7
1.30	(2.4)	-	-	7/7	7/7
	(1)	7/7	7/7	-	-
0.712	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

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

                          (1)       = Normal behaviour                          

                          (2.1)    = Lethargy

                          (2.2)    = Fish lying on its side

                          (2.4)    = Slowed escape reflex                    

                          (2.5)    = Missing escape reflex

                          (4.1)    = Hyperventilation

                          (E)       = Exitus lethalis

                          -           = No observation

                ---            = No observation due to 100 % mortality

Cumulative Mortality [%] in the Test Vessels

Geometric mean measured test item concentration [mg/L]	Cumulative mortality at observation time [hours]
	24	48	72	96
5.85	100	100	100	100
3.74	0	29	100	100
2.46	0	0	0	0
1.30	0	0	0	0
0.712	0	0	0	0
Control	0	0	0	0

Measured Concentrations and Percent of nominal concentration of 3-(Isodecyloxy)propionitrile

Sampling date	2013-03-04 Start of exposure (0 h)		2013-03-05 old medium (24 h)		2013-03-07 new medium (72 h)		2013-03-08 End of exposure (96 h)
Start of analysis	2013-03-04		2013-03-05		2013-03-07		2013-03-08
Nominal concentration	3-(Isodecyloxy)propionitrile
Test item [mg/L]	Meas. conc.  [mg/L]^1)	%	Meas. conc.  [mg/L]	%	Meas. conc.  [mg/L]	%	Meas. conc.  [mg/L]	%	Geometric mean meas. conc.
									[mg/L]	%
10.0	8.75	88	3.91	39	-		-		5.85	59
5.64	4.97	88	2.81	50	-		-		3.74	66
3.17	2.66	84	2.06	65	2.83	89	2.35	74	2.46	78
1.77	1.57	89	1.11	63	1.54	87	1.08	61	1.30	73
1.00	0.943	94	0.568	57	0.967	97	0.497	50	0.712	71
Control	LOQM		LOQM		LOQM		LOQM		LOQM

Meas. conc. = Measured concentration of the test item, mean value of 2 injections, dilution factor taken into account

%                   = Percent of the nominal concentration of the test item

LOQ_M            = Limit of quantification of the analytical method (50.0 µg/L)

-                     = No measurement due to 100 % mortality

¹⁾                              = reanalysed on 2013-03-05, mean value of 4 injections

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item was found to be toxic to zebrafish with a LC50-value of 3.03 (2.46 - 3.74) mg/L after 96 hours. At the geometric mean measured test item concentration of 0.712 mg/L no mortality or non lethal effects were observed (NOEC). The geometric mean measured concentration of 1.30 mg/L was the lowest concentration (LOEC) where effects (non-lethal) were observed. All effect levels are given based on the geometric mean measured concentrations of the test item 3-(Isodecyloxy)propionitrile.

Executive summary:

The acute toxicity of the test item 3-(Isodecyloxy)propionitrile(batch no.: F-212252)to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from 2013-03-04 to 2013-03-08 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test with daily renewal of the test media was performed with nominal test item concentrations of 1.00 – 1.77 – 3.17 – 5.64 – 10.0 mg/L, corresponding to geometric mean measured test item concentrations of 0.712 – 1.30 – 2.46 – 3.74 – 5.85 mg/L. Duration of the test was 96 hours. 7 test organisms were exposed to each concentration and the control. Water quality parameters pH-value, temperature and oxygen-saturation, measured after 0, 24, 48, 72 and 96 h were determined to be within the acceptable limits.

The concentrations of the test item 3-(Isodecyloxy)propionitrilewere analytically verified by LC-MS/MS from freshly prepared media after 0 h and 72 h and from corresponding 24 h aged test media after 24 h and 96 h.

The measured concentrations of the test item in fresh media (0 h and 72 h) were in the range of 84 to 97 % of the nominal values. The measured concentrations of the test item in old media (24 h and 96 h) were in the range of 39 to 74 % of the nominal values. Therefore, all effect levels are given based on the geometric mean measured concentrations of the test item.

LC-Values (24 – 96 hours)

Based on geometrical mean measured test item concentrations [mg/L].

Test duration [hours]	LC10	LC20	LC50
24	3.74 LC10 5.85	3.74 LC20 5.85	4.68 (3.74 - 5.85)
48	3.65 (3.59 – 3.69)*	3.71 (3.67 – 3.72)*	3.80 (3.78 – 3.87)*
72	2.46 LC10 3.74	2.46 LC20 3.74	3.03 (2.46 - 3.74)
96	2.46 LC10 3.74	2.46 LC20 3.74	3.03 (2.46 - 3.74)
LC0 = Highest test item concentration with 0 % mortality after 96 hours	2.46
LC100 = Lowest test item concentration with 100 % mortality after 96 hours	3.74

* 95 % confidence interval