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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-11-06 to 2013-11-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, no deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
0.0444 – 0.0888 – 0.178 – 0.355 – 0.710 mg/L (factor 2.0)

The test concentrations were based on the results of a preliminary range finding test

Overall Survival and Mortality in the 1st Preliminary Test on Study Day 2 (non GLP)
(static; 30 eggs per concentration)

Nominal Survival of eggs [n] Overall survival [%] Overall mortality [%]
test item per Study Day
concentration
[mg/L] 1 2
7.10 0 0 0 100
0.710 11 0 0 100
Control 30 30 100 0


Overall Survival and Mortality in the 2nd Preliminary Test on Study Day 7 (non GLP)
(semi-static; 30 eggs per concentration)

Nominal Survival of eggs/larvae [n] Overall survival [%] Overall mortality [%]
test item per Study Day
concentration
[mg/L] 2 3 5 7
0.710 17 16 0 --- 0 100
0.0710 29 29 29 29 97 3
Control 29 29 29 29 97 3


- Sampling method: Analytical evaluation of the various concentrations of C18/C18 unsatd.-Glucamide and the control was carried out via LC-MS/MS. Samples were taken from alternating test replicates in 3 sampling intervals from freshly prepared and corresponding aged test solutions.

The sorption of the test item on glass was quantified. Two separate replicates (without eggs) were set up for two sampling intervals for all test concentrations (1 replicate per test concentration per sampling day). These replicates were treated and incubated as test replicates until sampling.
After sampling for the test medium analysis (aqueous phase), the test vessels were rinsed with an appropriate volume of acetonitrile containing 0.2 % formic acid. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Test item, which was dissolved in remaining test medium adhering to the glassware, was subtracted by weighing. Therefore, the glassware was weighed before use and after being emptied. The difference in weight corresponds to the remaining test medium.

Schedule of Specific Test Item Analysis

Study Day 0 2 2 5 5 7
Fresh media Old media Fresh media Old media Fresh media Old media
Appropriate
dosage
level(s) and
control aqueous aqueous phase aqueous aqueous aqueous aqueous phase + glass surface
phase + glass surface phase phase phase

- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1.00 mg/L was freshly prepared with dilution water. Other test concentrations were prepared from this stock solution by dilution.

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.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbundesamt,
Schichauweg 58, 12307 Berlin, Germany)
- Method of breeding: A breeding stock of unexposed, mature zebrafish with an age of 12 months was used for 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 (0.1 - 10 µmol photons * m-2 * s-1on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum;
dry food sera vipan SERA GMBH, ad libitum.
- No disease treatments were administered
- 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

- Spawning: Adult zebrafish were kept in separate aquaria. About 15 minutes before start of artificial dawning rectangular dishes (26 cm x 14 cm x
6 cm), covered with a stainless steel mesh and provided with artificial plants, were introduced into the aquaria. After approximately 1 hour the
glass dishes were gently removed. A sufficient number of eggs was taken, washed in dilution water and immediately transferred into vessels
containing the respective exposure solutions without regard to fertilization (start of exposure). Eggs were fully covered with the respective test
solutions.

- Fertilization check: After approximately 2 h post fertilization, eggs were checked for fertilization. Under a stereo microscope every embryo was
checked for its blastomer phase. Eggs with only a 2 cell blastomer were regarded not to be fertilised. These eggs as well as coagulated eggs were
discarded.

- Introduction of eggs: Only fertilised eggs with more than 2 cells were introduced in the test vessels. 10 eggs were introduced per replicate
(corresponding to 30 eggs per treatment group).

- Feeding during test: no feeding was provided during the test. The sac-fry was nourished from the yolk-sac until end of exposure
(5 days post-hatch).

ACCLIMATION
- Acclimation not neccessary as breeding conditions are the same as test conditions

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 d

Post exposure observation period:

5 days post hatch

Hardness:

The total hardness, measured at the beginning of the exposure from one replicate per test concentration and control, was in the range of 50 – 53 mg CaCO3/L.

Test temperature:

The mean water temperature measured every renewal interval from freshly prepared and aged test solutions during exposure period in alternating replicates
of the control was 25.3 °C and ranged from a minimum temperature of 25.0 °C to a maximum temperature of 25.7 °C.

The temperature of the dilution water was measured continuously once per hour throughout exposure. The mean temperature  mean standard deviation was 25.3  0.3 °C

Water Temperature in the Test Media

Study day Test media Water temperature [°C]
Nominal test item concentration [mg/L]
Control 0.0444 0.0888 0.178 0.355 0.710
0 new 25.0 25.0 25.0 25.0 25.1 25.1
2 old 24.7 24.8 24.9 25.0 25.0 24.9
new 24.8 24.8 24.8 24.9 24.8 25.0
5 old 24.8 24.8 24.8 24.9 25.0 25.0
new 25.2 25.3 25.2 25.3 25.3 25.2
7 old 25.2 25.1 25.1 25.2 25.2 25.2
new 25.5 25.3 25.3 25.1 25.1 25.1
9 old 24.5 24.4 24.4 24.4 24.4 24.3
Mean 25.0 24.9 24.9 25.0 25.0 25.0
SD ± 0.32 0.30 0.28 0.27 0.28 0.29
Min. 24.5 24.4 24.4 24.4 24.4 24.3
Max. 25.5 25.3 25.3 25.3 25.3 25.2



Water Temperature (Continuous Measuring) in the Dilution Water:

Period of measurements 2014-01-22 to 2014-01-31
Minimum temperature [°C] 24.3
Maximum temperature [°C] 26.0
Mean temperature
± Standard deviation [°C] 25.3 ± 0.3

pH:

The pH-values in the control and test item groups ranged from 7.02 to 7.47 and from 7.08 to 7.61 during exposure, respectively.


pH Values in the Test Media:

Study day Test media pH-value
Nominal test item concentration [mg/L]
Control 0.0444 0.0888 0.178 0.355 0.710
0 new 7.02 7.08 7.18 7.12 7.16 7.19
2 old 7.11 7.20 7.27 7.30 7.32 7.28
new 7.18 7.21 7.27 7.28 7.28 7.28
5 old 7.47 7.53 7.55 7.58 7.61 7.60
new 7.14 7.28 7.34 7.38 7.42 7.43
7 old 7.12 7.18 7.27 7.26 7.40 7.45
new 7.17 7.18 7.29 7.31 7.32 7.35
9 old 7.12 7.20 7.27 7.32 7.34 7.40
Mean 7.17 7.23 7.31 7.32 7.36 7.37
SD ± 0.13 0.13 0.11 0.13 0.13 0.13
Min. 7.02 7.08 7.18 7.12 7.16 7.19
Max. 7.47 7.53 7.55 7.58 7.61 7.60

Dissolved oxygen:

The dissolved oxygen concentrations in the control and test item groups, expressed in percent saturation, were in the range of 94 – 100 % during exposure.


Dissolved Oxygen in Percent Air Saturation Value:

Study day Test media Dissolved Oxygen [%]
Nominal test item concentration [mg/L]
Control 0.0444 0.0888 0.178 0.355 0.710
0 new 100 100 100 100 100 100
2 old 98 99 100 99 100 100
new 99 98 99 99 99 99
5 old 98 98 97 98 98 97
new 100 100 100 99 99 99
7 old 98 99 98 98 97 96
new 99 100 100 100 99 99
9 old 94 99 98 99 97 99
Mean 98 99 99 99 99 99
SD ± 1.91 0.83 1.20 0.76 1.19 1.41
Min. 94 98 97 98 97 96
Max. 100 100 100 100 100 100

Nominal and measured concentrations:

C18/C18 unsatd.-Glucamide is an UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in water at room temperature. All effect concentrations are given as nominal concentrations of the test item and the main components (sum of glucamides). The concentrations of the unsatd.C18 fraction of the test item C18/C18 unsatd.-Glucamide were analytically verified by LC-MS/MS from alternating test replicates in 3 sampling intervals from freshly prepared (days 0, 2 and 5) and corresponding aged test media (days 2, 5 and 7) at all concentration levels and the control. The measured concentrations of the unsatd.C18 fraction of the test item C18/C18 unsatd.-Glucamide in freshly prepared test media (days 0, 2 and 5) were in the range of 65 to 111 % of the nominal values. In corresponding aged test media (days 2, 5 and 7) the measured concentrations of the unsatd.C18 fraction of the test item C18/C18 unsatd.-Glucamide were in the range of range of below the limit of quantification of the analytical method (LOQM = 2.23 µg unsatd.C18 fraction/L) to 83 % the nominal values.

Details on test conditions:

TEST SYSTEM
- Test vessel: Crystallisation dishes (inner diameter 13.5 cm, water height about 5 cm), Volume of the test media = approximately 500 mL
- Type (delete if not applicable): test vessel closed with a lid (with 1 hole, diameter approximately 1 cm)
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): semi-static test procedure with renewal of the test media every 2 to 3 days; for the renewal of
the test media the test organisms were retained in the test vessels whilst a proportion of at least 75 % of the test medium was changed. Freshly
prepared media was gently added to test vessels to avoid stressing of the test organisms.
- No. of organisms per vessel: 10 eggs per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin; the water was filtered on activated charcoal and aerated for at least 24 h to
remove chlorine; Total hardness: 10 - 250 mg CaCO3/L, pH-value: 6.0 - 8.5
- Total organic carbon: The total organic carbon of the dilution water (TOC, measured as NPOC) determined at the beginning of the exposure was
1.45 mg/L.
- Residual Chlorine: Residual chlorine of the dilution water, determined at the beginning of the exposure was ≤ 0.010 mg/L.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, pH value and oxygen saturation were measured on each water renewal interval from freshly
prepared and old test media in one replicate per test concentration and the control, respectively. Water temperature was recorded continuously by
a datalogger in a surrogate test vessel filled with dilution water. Total hardness was measured at the beginning of the exposure from one replicate
per test concentration and control, respectively. Chlorine and TOC were measured at the beginning of the test from the dilution water.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 h photoperiod (light/dark)
- Light intensity: Light intensity was measured at start of exposure on the surface of the test vessels and ranged from 3.78 to 4.84 µmol photons *
m-2 * s-1 (mean: 4.29 µmol photons * m-2 *s-1). The test vessels were positioned randomly and repositioned daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Test concentrations: Nominal test concentrations: 2.50 – 5.00 – 10.0 – 20.0 – 40.0 mg/L

Reference substance (positive control):

not required

Key result

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: overall survival

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Duration:

9 d

Dose descriptor:

LC50

Effect conc.:

0.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CI (0.46 -> 0.71 mg/L)

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

0.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight

Details on results:

- Egg Fertilization Rate: The egg fertilization rate, determined on study day 0 (start of exposure) was ≥ 98 %.
- Egg Hatch and Definition of Post Hatch Day 0: Egg hatch began on study day 3 in the control and the test concentrations of 0.0444 to 0.355 mg/L and continued until study day 5. Egg hatch in the highest test concentration began on study day 4 and continued until study day 5. Study day 4 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 100 %.
Statistical procedures (One Way Analysis of Variance) were applied for study days 3, 4 and 5 (post hatch days -1, 0 and 1). Statistically significant differences were found for post hatch day 0 and 1 for the highest concentration level of 0.710 mg/L.


Egg Hatch / Hatching Time:

Nominal test
item Egg hatch [%]
concentration Replicate
[mg/L] Post hatch day -2 Post hatch day -1 Post hatch day 0 Post hatch day 1 Post hatch day 2
(Study day 2) (Study day 3) (Study day 4) (Study day 5) (Study day 6)

0.710 1 0 0 20 20 20
2 0 0 50 50 50
3 0 0 20 60 60
Mean 0 (-) 0 (+) 30 (+) 43 43
0.355 1 0 50 100 100 100
2 0 20 100 100 100
3 0 10 100 100 100
Mean 0 (-) 27 (-) 100 (-) 100 100
0.178 1 0 90 90 90 90
2 0 40 100 100 100
3 0 40 100 100 100
Mean 0 (-) 57 (-) 97 (-) 97 97
0.0888 1 0 80 100 100 100
2 0 40 90 90 90
3 0 40 100 100 100
Mean 0 (-) 53 (-) 97 (-) 97 97
0.0444 1 0 90 100 100 100
2 0 30 100 100 100
3 0 40 100 100 100
Mean 0 (-) 53 (-) 100 (-) 100 100
Control 1 0 40 100 100 100
2 0 10 100 100 100
3 0 20 100 100 100
Mean 0 (-) 23 (-) 100 (-) 100 100


(+) = Statistically significant difference from control (=0.05, ANOVA)
(-) = No statistically significant difference from control (=0.05, ANOVA)


- Swim-up: Swim-up was observed for a 3-day period on study days 5 to 7. Newly hatched fry of the control and the test concentrations of 0.0444 to 0.355 mg/L began to swim up on study day 5 (post hatch day 1). On study day 6 (post hatch day 2) all surviving larvae of the control and the test concentrations of 0.0444 to 0.355 mg/L had swum up. Newly hatched fry of the highest test concentration of 0.710 mg/L began to swim up on study day 6 (post hatch day 2). On study day 7 (post hatch day 3) all surviving larvae of this test concentration had swum up. No significance tests were carried out for swim-up.

Percent Swim-up of Hatched Fry:

Nominal test item
concentration
[mg/L] Replicate Swim-up [%]

PHD 0 PHD 1 PHD 2 PHD 3
(Study day 4) (Study day 5) (Study day 6) (Study day 7)
0.710 1 0 0 0 100
2 0 0 40 100
3 0 0 67 100
Mean 0 0 36 100
0.355 1 0 100 100 100
2 0 100 100 100
3 0 60 100 100
Mean 0 87 100 100
0.178 1 0 89 100 100
2 0 90 100 100
3 0 80 100 100
Mean 0 86 100 100
0.0888 1 0 90 100 100
2 0 89 100 100
3 0 70 100 100
Mean 0 83 100 100
0.0444 1 0 100 100 100
2 0 60 100 100
3 0 80 100 100
Mean 0 80 100 100
Control 1 0 70 100 100
2 0 70 100 100
3 0 30 100 100
Mean 0 57 100 100



- Fry Survival (Post Hatch Survival) : The post hatch success in all control replicates met the guideline criteria. The post hatch survival was calculated from the study day with the maximum hatch rate and the lowest count of remaining vital eggs per test group (post hatch day 0: control and 0.0444 to 0.355 mg/L; post hatch day 1: 0.710 mg/L) and the surviving larvae on post hatch day 5. The post hatch survival at the end of the study was 100 % in the control group and ranged from 93 to 100 % post hatch success in the tested concentration levels, respectively.
One Way Analysis of Variance and DUNNETT’S test were carried out for post hatch survival at the end of the study. No statistically significant differences were found for the tested concentrations when compared with the control.



Post Hatch Survival on Study Day 9 (Post Hatch Day 5):

Nominal
test item
concentration
[mg/L] Study day Replicate Hatched larvae on Vital larvae on Post hatch
with maximum hatch study day with study day 9 survival
hatch maximum hatch (PHD 5) [%]
n/10 n/10

0.710 5
(PHD 1) 1 2 2 100
2 5 4 80
3 6 6 100
Mean 4.3 4.0 (-) 93
0.355 4
(PHD 0) 1 10 10 100
2 10 9 90
3 10 10 100
Mean 10.0 9.7 (-) 97
0.178 4
(PHD 0) 1 9 9 100
2 10 10 100
3 10 10 100
Mean 9.7 9.7 (-) 100
0.0888 4
(PHD 0) 1 10 10 100
2 9 9 100
3 10 10 100
Mean 9.7 9.7 (-) 100
0.0444 4
(PHD 0) 1 10 10 100
2 10 10 100
3 10 10 100
Mean 10.0 10.0 (-) 100
Control 4
(PHD 0) 1 10 10 100
2 10 10 100
3 10 10 100
Mean 10.0 10.0 (-) 100


(-) = No statistically significant difference from control (=0.05, ANOVA)


- Overall Survival: Overall survival at the end of the study was 100 % in the control group and ranged from 40 to 100 % in the tested concentration levels, respectively.
One Way Analysis of Variance and DUNNETT’S test were carried out for the results of overall survival on post hatch day 5 (end of the study). A statistically significant difference was found for the highest test concentration of 0.710 mg/L when compared with the control.



Overall Survival and Mortality on Study Day 9 (Post Hatch Day 5):

Nominal
test item
concentration
[mg/L] Replicate Live fry on Overall survival Mortality
post hatch day 5 [%] [%]
(study day 9)
n/10

0.710 1 2 20 80
2 4 40 60
3 6 60 40
Mean 4 (+) 40 60
0.355 1 10 100 0
2 9 90 10
3 10 100 0
Mean 10 (-) 97 3
0.178 1 9 90 10
2 10 100 0
3 10 100 0
Mean 10 (-) 97 3
0.0888 1 10 100 0
2 9 90 10
3 10 100 0
Mean 10 (-) 97 3
0.0444 1 10 100 0
2 10 100 0
3 10 100 0
Mean 10 (-) 100 0
Control 1 10 100 0
2 10 100 0
3 10 100 0
Mean 10 (-) 100 0



(-) = No statistically significant difference from control (=0.05, ANOVA)



- Fry Growth: The fry growth, expressed as length and dry weight, was determined on study day 9 (post hatch day 5).
One Way Analysis of Variance and DUNNETT’S test were carried out for the results of post hatch day 5 (end of the study). For the weight and length data no statistically significant differences were found for the tested concentrations, respectively.



Fry Growth: Length and Dry Weight on Study Day 9 (Post Hatch Day 5):

Post hatch day 5 (study termination)

Nominal test item concentration Replicate Number Mean length Pooled Mean dry weight per concentration of per fish larvae dry weight fish larvae
[mg/L] larvae [mm] [mg] [mg]

0.710 1 2 3.817 0.200* 0.033*
2 4 3.847
3 6 3.704 0.188 0.031
Mean (-) 3.789 0.194 (-) 0.032
0.355 1 10 3.908 0.358 0.036
2 9 3.762 0.300 0.033
3 10 3.779 0.320 0.032
Mean (-) 3.816 0.326 (-) 0.034
0.178 1 9 3.754 0.274 0.030
2 10 3.789 0.261 0.026
3 10 3.784 0.256 0.026
Mean (-) 3.776 0.264 (-) 0.027
0.0888 1 10 3.695 0.340 0.034
2 9 3.763 0.338 0.038
3 10 3.747 0.348 0.035
Mean (-) 3.735 0.342 (-) 0.036
0.0444 1 10 3.673 0.310 0.031
2 10 3.780 0.350 0.035
3 10 3.804 0.338 0.034
Mean (-) 3.752 0.333 (-) 0.033
Control 1 10 3.783 0.332 0.033
2 10 3.682 0.276 0.028
3 10 3.752 0.356 0.036
Mean (-) 3.739 0.321 (-) 0.032


* fish larvae of replicate 1 and 2 were pooled for determination of dry weight

(-) = No statistically significant difference from control (=0.05, ANOVA)


- Morphological and Behavioural Observations: On study day 5 and 6 some larvae of the concentration level 0.710 mg/L showed side position and oedema. From study days 6 to 9 (end of the study) some larvae of the concentration level 0.710 mg/L showed quiescence marked by abnormally low activity. Other larvae of the control group and the test concentrations of 0.0444 to 0.355 mg/L showed no morphological or behavioural abnormalities.
Fisher’s Exact Test (two sided) was carried out for the observations of behavioural abnormalities (quiescence) on study day 9 (PHD 5). For the non-lethal observations of the concentration level of 0.710 mg/L statistically significant differences were found.


Morphological and Behavioural Observations:

Nominal Rep. Observation* Number of vital larvae affected at observation time
test item conc.
[mg/L]

PHD 1 PHD 2 PHD 3 PHD 4 PHD 5
(Study day 5) (Study day 6) (Study day 7) (Study day 8) (Study day 9)
0.710 1 (N) - - - - -
(Q) - 2 2 2 (+) 2
(S) - 2 - - -
(O) 2 2 - - -
2 (N) 5 2 - - -
(Q) - 3 5 5 (+) 4
(S) - 3 - - -
(O) - - - - -
3 (N) 6 4 - - -
(Q) - 2 6 6 (+) 6
(S) - - - - -
(O) - - - - -
0.355 1 - 3 (N)
0.178 1 - 3 (N)
0.0888 1 - 3 (N) All remaining vital larvae
0.0444 1 - 3 (N)
Control 1 - 3 (N)



(N) = Normal appearance and behaviour
(S) = Scoliosis
(O) = Oedema (Q) = Quiescence
- = No observations

(+) = Statistically significant difference from control (Fisher’s Exact Test, two sided)

Reported statistics and error estimates:

One way analysis of variance (ANOVA) and DUNNETT’S test was used for NOEC/LOEC calculations. When running a one way analysis of variance a normality test and an equal variance test were done first.
For the parameters hatch, post hatch survival and overall fry survival (mortality), growth (length and dry weight), the following statistical tests were conducted:
Hatching data of study days 3 to 5 (post hatch days -1 to 1) were analysed with ANOVA. DUNNETT’S test was used for NOEC/LOEC calculation of hatching data of study days 4 and 5. Normality tests failed for hatch data of study days 3 to 5. No transformation was carried out with these data because the data set was not estimated to follow a normal distribution.
Post hatch survival and overall survival data (mortality), dry weight and length data of study day 9 (post hatch day 5) were analysed with ANOVA. DUNNETT’S test was used for NOEC/LOEC calculation of overall survival and dry weight data of study day 9, respectively. Normality tests failed for post hatch and overall survival data, respectively. No transformation was carried out with these data because the data set was not estimated to follow a normal distribution.
Behavioural effects (quiescence), observed on study day 9, were analysed with Fisher’s exact test (two sided), as revised in OECD Series on Testing and Assessment No. 54 (2006).
The statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The -value (acceptable probability of incorrectly concluding that there is a difference) was 0.05.


The LC50-value and the corresponding confidence interval after 5 days post-hatch were calculated by standard procedures (sigmoidal dose-response regression) as revised in OECD Series on Testing and Assessment No. 54 (2006).

Validity criteria fulfilled:

yes

Conclusions:

C18/C18 unsatd.-Glucamide caused significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under semi-static conditions at the dosage level of 0.71 mg/L (nominal test item concentration). The LC50 on study day 9 (post hatch day 5) was determined to be 0.67 (0.46 – > 0.71) mg/L. The NOEC for the parameters hatch and overall survival (mortality) was 0.36 mg/L. The NOEC for the post hatch survival and growth (expressed as length and weight) was 0.71 mg/L.
All effect levels given are based on the nominal concentrations of the test item C18/C18 unsatd.-Glucamide and the main component (sum of Glucamides).

Executive summary:

The effects of the test item C18/C18 unsatd.-Glucamide (batch no.:SN119/13/3-8) to the embryo and sac-fry stages of fish (Zebrafish /Danio rerio) were determined according to OECD Guideline 212 from 2014‑01‑22 to 2014-02-03 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test procedure with renewal of the test media every 2 to 3 days was performed with the nominal test item concentrations of 0.0444– 0.0888 – 0.178 – 0.355 – 0.710 mg/L (factor 2.0).

 

The test was started by placing fertilized eggs in the test vessels and lasted 9 days (5 days post-hatch). 30 eggs of Danio rerio were exposed per test concentration and control (3 replicates with 10 eggs each), respectively.

 

On day four 100 % of the control larvae have hatched. Therefore, study day 4 was defined as post hatch day 0 (= PHD 0).

 

Different toxic endpoints were determined: egg hatch, time to hatch, post hatch survival, overall fry survival, mortality and fry growth (expressed as length and weight), respectively. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and the LC₅₀-value were determined based on the statistical results.

 

C18/C18 unsatd.-Glucamide is an UVCB substance (Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials) and is poorly soluble in water at room temperature. All effect concentrations are given as nominal concentrations of the test item and the main components (sum of glucamides).

The concentrations of the unsatd. C18 fraction of the test itemwereanalytically verified via LC-MS/MS in freshly prepared test media (days 0, 2 and 5) and in corresponding aged test media (days 2, 5 and 7) in all concentration levels and the control.

The measured concentrations of the unsatd. C18 fraction of the test item in freshly prepared test media (days 0, 2 and 5) were in the range of 65 to 111 % of the nominal values. In corresponding aged test media (days 2, 5 and 7) the measured concentrations of the unsatd. C18 fraction of the test item were in the range of below the limit of quantification of the analytical method (LOQ_M= 2.23 µg C18 unsatd.-Glucamide/L) to 83 % of the nominal values.

Adsorption to the glass walls of the test vessels was determined at the end of two sampling intervals (days 2 and 7) from additional replicates (prepared without eggs). Adsorption to the glass walls in these replicates was in the range of below LOQ_Mto 17 % of the nominal concentrations of the unsatd. C18 fraction of the test item.

 

The effect levels given are based on the nominal concentrations of the test item C18/C18 unsatd.-Glucamide and the main components (sum of Glucamides).

NOEC, LOEC: Hatch, Fry Survival and Fry Growth

Based on nominal concentrations of the test itemC18/C18 unsatd.-Glucamide and

the main component (Sum of Glucamides) [mg/L].

Parameter	C18/C18 unsatd.-Glucamide [mg/L]		Main components (Sum of Glucamides, 80.4 % w/w) [mg/L]
	NOEC	LOEC	NOEC	LOEC
Hatch	0.355	0.710	0.285	0.571
Post hatch survival	0.710	> 0.710	0.571	> 0.571
Overall survival	0.355	0.710	0.285	0.571
Length	0.710	> 0.710	0.571	> 0.571
Dry weight	0.710	> 0.710	0.571	> 0.571

LC₅₀-Value with 95 % Confidence Interval on Study Day 9 (Post Hatch Day 5)

               Based on nominal concentrations of the test item C18/C18 unsatd.-Glucamide and

               the main component (Sum of Glucamides) [mg/L].

 

	C18/C18 unsatd.-Glucamide [mg/L]	Main components (Sum of Glucamides, 80.4 % w/w) [mg/L]
LC50	0.669	0.538
95 % confidence interval	0.457 - > 0.710	0.367 - > 0.571

Description of key information

C18/C18 unsatd.-Glucamide caused significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under semi-static conditions at the dosage level of 0.71 mg/L (nominal test item concentration). The LC50 on study day 9 (post hatch day 5) was determined to be 0.67 (0.46 – > 0.71) mg/L. The NOEC for the parameters hatch and overall survival (mortality) was 0.36 mg/L. The NOEC for the post hatch survival and growth (expressed as length and weight) was 0.71 mg/L. All effect levels given are based on the nominal concentrations of the test item C18/C18 unsatd.-Glucamide and the main component (sum of Glucamides).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.36 mg/L