D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

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:

2013-12-16 to 2013-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according GLP, no deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.444 - 0.888 - 1.78 - 3.55 - 7.10 mg/L
The test concentrations are based on the results of two preliminary range finding tests.
- Sampling method:
Analytical evaluation of the various concentrations of C18/C18 unsatd.-Glucamide and the control was carried out via LC-MS/MS from freshly prepared media after 0 and 72 h and from corresponding aged test media after 24 and 96 h or whenever 100 % mortality occured.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing. The test media were mixed with an ultraturrax (1 min, 17000 rpm).

- Eluate: Dilution water
- Differential loadings: 0.444 - 0.888 - 1.78 - 3.55 - 7.10 mg/L (nominal, factor 2)
- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

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: 2.9 cm
- Weight at study initiation (mean and range, SD): Average body weight: 0.30 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

Total Hardness at day 0: 54 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 incl. tables"

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, 2.5 L test volume per vessel
- Aeration: Gentle aeration
- Renewal rate of test solution (frequency/flow rate): semi-static design with 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
- Biomass loading rate: 0.83 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all vessels at the start and end of the exposure and every 24 hours (from freshly prepared and 24 h aged test media). Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at test start.


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 studies:

Cumulative Mortality [%] in the first Preliminary Test (non GLP)
(static, n = 3)
Nominal Test Item Concentration
[mg/L] Cumulative mortality [%] after different exposure periods [hours]
24 48 72 96
1.00 0 0 0 0
0.100 0 0 0 0
Control 0 0 0 0


Cumulative Mortality [%] in the second Preliminary Test (non GLP)
(semi-static, n = 4)
Nominal Test Item Concentration
[mg/L] Cumulative mortality [%] after different exposure periods [hours]
24 48 72 96
7.10 100 --- --- ---
1.00 0 0 0 0
Control 0 0 0 0

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI (1.78-2.55 mg/L)

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): The test concentrations were clearly solved throughout exposure. Except 24 hours aged test media of the nominal test concentrations of 3.55 and 7.10 mg/L (test replicates with fish), where turbidity was caused by microbial degradation of dead fish (100 % mortality).

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

The LC50-value after 24 hours of exposure was calculated by straight line regression. LC10,20-values could not be estimated since only one single concentration level gave partial response thus were given as a range of concentrations causing 0 and 100 % mortality.
After 48 to 96 hours only 0 and 100 % mortality occurred. Hence the LC50 after 48 to 96 hours is the geometric mean value of the nominal 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 were not applicable and were given as a range of those concentrations. The chosen methods for 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 test results.

Sublethal observations / clinical signs:

Observations in the Test Vessels

Nominal test item concentration [mg/L]	Observation *)	Effects on behaviour after different exposure periods [hours]
		24	48	72	96
7.10	(E)	7/7	---	---	---
3.55	(E)	4/7	3/3	---	---
	(2.7)	3/7	-	---	---
	(2.6)	3/7	-	---	---
1.78	(2.8)	-	5/7	5/7	5/7
	(2.7)	7/7	7/7	7/7	7/7
	(2.6)	7/7	7/7	7/7	7/7
0.888	(1)	7/7	7/7	7/7	7/7
0.444	(1)	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7

Cumulative Mortality [%] in the Test Vessels

Nominal test item concentration [mg/L]	Cumulative mortality [%] after different exposure periods [hours]
	24	48	72	96
7.10	100	100	100	100
3.55	57	100	100	100
1.78	0	0	0	0
0.888	0	0	0	0
0.444	0	0	0	0
Control	0	0	0	0

Measured Concentrations and Percent of Nominal Concentrations of theunsatd.C18fractionof the test itemC18/C18 unsatd.-GlucamideGlass Walls of Test Vessels without Fish after 24 and 96 hours

 

Sampling date		2013-12-17 Adsorption to glass (24 h)		2013-12-20 Adsorption to glass  (96 h)
Start of analysis		2013-12-17		2013-12-20
Nominal concentration [mg/L]		Unsatd. C18 fraction of the test itemC18/C18 unsatd.-Glucamide
C18/C18 unsatd.-Glucamide (Test item)	Unsatd.C18 unsatd.-Glucamide	Meas. conc.  [mg/L]	%	Meas. conc.  [mg/L]	%
7.10	5.28	0.177	3	---
3.55	2.64	0.172	7	---
1.78	1.32	0.116	9	0.133	10
0.888	0.660	0.0521	8	0.0631	10
0.444	0.330	0.0285	9	0.0341	10
Control		< LOQM		< LOQM

Meas. conc.    measured concentrationofC18 unsatd.-Glucamideof the test item

                        C18/C18 unsatd.-Glucamide, mean value of 2 injections, dilution and enrichment factor taken into account

%                  = Percent of the nominal concentration of the unsatd. C18 fraction of the test item

LOQ_M          = Limit of quantification of theanalytical method(2.23 µg/L of the unsatd. C18 fraction of the test item)

---                 = No measurement due to 100 % mortality

Measured Concentrations and Percent of Nominal Concentrations ofunsatd.C18 fraction of the test item C18/C18 unsatd.-Glucamide Glass Walls of Test Vessels with Fish after 24 and 96 hours

 

Sampling date		2013-12-17 Adsorption to glass (24 h)		2013-12-20 Adsorption to glass  (96 h)
Start of analysis		2013-12-17		2013-12-20
Nominal concentration [mg/L]		Unsatd. C18 fraction of the test itemC18/C18 unsatd.-Glucamide
C18/C18 unsatd.-Glucamide (Test item)	Unsatd.C18 unsatd.-Glucamide	Meas. conc.  [mg/L]	%	Meas. conc.  [mg/L]	%
7.10	5.28	0.177	3	---
3.55	2.64	0.104	4	---
1.78	1.32	0.175	13	0.099	8
0.888	0.660	0.0468	7	0.0536	8
0.444	0.330	0.00899	3	0.0283	9
Control		< LOQM		< LOQM

Meas. conc.    measured concentrationofC18 unsatd.-Glucamideof the test item

                        C18/C18 unsatd.-Glucamide, mean value of 2 injections, dilution and enrichment factor taken into account

%                  = Percent of the nominal concentration of the unsatd. C18 fraction of the test item

LOQ_M          = Limit of quantification of theanalytical method(2.23 µg/L of the unsatd. C18 fraction of the test item)

---                 = No measurement due to 100 % mortality

Validity criteria fulfilled:

yes

Conclusions:

In this study C18/C18 unsatd.-Glucamide was found to cause lethal effects to Zebrafish after 96 hours at the nominal test item 3.55 mg/L and higher. At the nominal test item concentration of 1.78 mg/L (1.43 mg/L main components (sum of Glucamides)) no mortality was observed. The LC50 (96h) is 2.51 mg/L (95% CI 1,78-2.55 mg/L).

All effect levels were given based on the nominal concentrations of the test item C18/C18 unsatd.-Glucamide and the main components (sum of Glucamides). The nominal LC50 of the test item after 96 hours was 2.51 (1.78 – 3.55) mg/L, corresponding to 2.02 (1.43 – 2.85) mg/L of the nominal concentrations of the main components (sum of Glucamides) (80.4 % w/w).

Executive summary:

The acute toxicity of the test item C18/C18 unsatd.-Glucamide (batch no.:SN119/13/3-8) to fish (zebrafish) was determined according to OECD-Guideline for Testing of Chemicals No. 203 (1992) from2013-12-16 to 2013-12-20 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 0.444 - 0.888 - 1.78 - 3.55 - 7.10 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.

 

C18/C18 unsatd.-Glucamideis an UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in water at room temperature.

The concentrations of unsatd. C18 fraction of the test item were analytically verified by LC-MS/MS in freshly prepared test media (0 h and 72 h) and in corresponding 24 hours aged test media (24 h and 96 h) in all concentration levels and the control.

The measured concentrations of the unsatd. C18 fraction in fresh media (0 h and 72 h) were in the range of 90 to 128 % of the nominal values. The measured concentrations of the unsatd. C18 fraction in old media (24 h and 96 h) were in the range of 14 to 84 % of the nominal values.

Adsorption to the glass walls of the test vessels was determined at the end of two sampling intervals (24 and 96 hours) from test replicates and additional replicates without fish. Adsorption to the glass walls in test replicates and additional replicates was in the range of 3 – 13 % of the nominal concentrations of the unsatd. C18 fraction.

 

The effect levels were given based on the nominal concentrations of the test itemC18/C18 unsatd.-Glucamideand the main components (sum of Glucamides).

 

LC-Values with 95 % Confidence Intervals (0 – 96 hours)

Based on nominal concentrations of the test itemC18/C18 unsatd.-Glucamide[mg/L].
Due to only partial response the LC₁₀and LC₂₀values were given as a range.
For the same reason no calculation of confidence intervals was carried out for the LC₅₀value.

Test duration [hours]	LC10(p = 95 %)	LC20(p = 95 %)	LC50(p = 95 %)
24	1.78 < LC10< 3.55	1.78 < LC20< 3.55	3.44 (1.78 – 7.10)
48	1.78 < LC10< 3.55	1.78 < LC20< 3.55	2.51 (1.78 – 3.55)
72	1.78 < LC10< 3.55	1.78 < LC20< 3.55	2.51 (1.78 – 3.55)
96	1.78 < LC10< 3.55	1.78 < LC20< 3.55	2.51 (1.78 – 3.55)
LC0= Highest test item concentration with 0 % mortality after 96 hours	1.78
LC100= Lowest test item concentration with 100 % mortality after 96 hours	3.55

 

LC-Values with 95 % Confidence Intervals (0 – 96 hours)

Based on nominal concentrations of the main components (sum of Glucamides)(80.4 % w/w) [mg/L].
Due to only partial response the LC₁₀and LC₂₀values were given as a range.
For the same reason no calculation of confidence intervals was carried out for the LC₅₀value.

Test duration [hours]	LC10(p = 95 %)	LC20(p = 95 %)	LC50(p = 95 %)
24	1.43 < LC10< 2.85	1.43 < LC20< 2.85	2.77 (1.43 – 5.71)
48	1.43 < LC10< 2.85	1.43 < LC20< 2.85	2.02 (1.43 – 2.85)
72	1.43 < LC10< 2.85	1.43 < LC20< 2.85	2.02 (1.43 – 2.85)
96	1.43 < LC10< 2.85	1.43 < LC20< 2.85	2.02 (1.43 – 2.85)
LC0= Highest test item concentration with 0 % mortality after 96 hours	1.43
LC100= Lowest test item concentration with 100 % mortality after 96 hours	2.85

Description of key information

In this study C18/C18 unsatd.-Glucamide was found to cause lethal effects to Zebrafish after 96 hours at the nominal test item 3.55 mg/L and higher. At the nominal test item concentration of 1.78 mg/L (1.43 mg/L main components (sum of Glucamides)) no mortality was observed. The LC50 (96h) is 2.51 mg/L (95% CI 1,78-2.55 mg/L). All effect levels were given based on the nominal concentrations of the test item C18/C18 unsatd.-Glucamide and the main components (sum of Glucamides). The nominal LC50 of the test item after 96 hours was 2.51 (1.78 – 3.55) mg/L, corresponding to 2.02 (1.43 – 2.85) mg/L of the nominal concentrations of the main components (sum of Glucamides) (80.4 % w/w).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2.51 mg/L