D-Glucitol, 1-deoxy-1-(methylamino)-, N-C8-10 acyl derivs.

Long-term toxicity to fish

Administrative data

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 10 09 to 2013-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed under GLP, no deficiencies

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-10(even numbered) acyl] derivs.

EC Number:

940-284-1

Cas Number:

1591782-62-5

Molecular formula:

C15H31NO6 C17H35NO6

IUPAC Name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-10(even numbered) acyl] derivs.

Constituent 2

Reference substance name:

Glucamide 810

IUPAC Name:

Glucamide 810

Details on test material:

- Physical state: Colourless powder
- Sum formula: C15H31NO6 (C8-Glucamide) C17H35NO6 (C10-Glucamide)
- Molecular weight:
C8-Glucamide: 321.42 g/Mol
C10-Glucamide 349.47 g/Mol
- Storage condition of test material: Room temperature, protected from light, in original container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 12.5 – 25.0 – 50.0 – 100 – 200 mg/L (factor 2.0)
- Sampling method: Analytical evaluation of the various concentrations of C8/C10-Glucamide AC93/13 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

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

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing. Agitation.
- Eluate: Dilution water
- Differential loading: 12.5 – 25.0 – 50.0 – 100 – 200 mg/L (factor 2.0)
- Controls: 30 eggs in dilution water (without test item, 3 replicates with 10 eggs each) will be 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
- Strain: Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Source: All fish eggs used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbuundesamt, Schichauweg 58, 12307 Berlin).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: 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.
- Subsequent handling of eggs: 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.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 d

Test conditions

Hardness:

The total hardness, measured at the beginning of the exposure from one replicate per test concentration and control, was in the range of 55 – 60 mg CaCO3/L.

Test temperature:

please refer to "Any other information on materials"

pH:

please refer to "Any other information on materials"

Dissolved oxygen:

please refer to "Any other information on materials"

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:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Crystallisation dishes (inner diameter 13.5 cm, water height about
5 cm) were used. The volume of the test media in the dishes was about 500 mL.
- Aeration: No aeration was provided. A semi-static test procedure with renewal of the test media every 2 to 3 days was performed. Eggs were exposed to the test item in crystallisation dishes filled with approximately 500 mL of the test media. 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 fertilized eggs/embryos per vessel: 10
- 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 was used. 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


OTHER TEST CONDITIONS
- pH: 6 – 8.5 within a range of 0.5 °C
- Photoperiod: 16 h photoperiod daily
- Light intensity: 0.1 - 10 µmol photons • m-2 • s-1on water surface (diffuse light)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Biological parameters Observations were made daily.

Hatched eggs The number of hatched eggs was determined daily until 5 days post-hatch.

Post hatch period Per definition the post hatch period begun when at least 80 % of all fertilized and living embryos in the control group have hatched (day 4 of the study).

Mortality Criteria for mortality vary according to life stage:

For eggs: Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, was checked daily. Dead eggs were discarded.

For embryos: Absence of body movement and/or heart-beat, change in coloration. Dead embryos were discarded.

For larvae: Immobility and/or absence of respiratory movement and/or absence of heart-beat (as far as visible) and/or lack of reaction to mechanical stimulus.

Further effects
Abnormal appearance and behaviour were also recorded.The number of larvae or fish showing abnormality of body form and/or pigmentation and the stage of yolk-sac absorption was recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour will also be recorded by visually inspecting each replicate.

Measurement of fish size Fish size was determined at the end of the exposure (post-hatch day 5) of the control and of three test groups where no significant mortalities were observed.
The fish were euthanized in a Benzocaine solution and the total length of each fish was measured to the nearest 0.001 mm with a microscope camera and corresponding software (ImageFocus, EUROMEX BV).



Measurement of
dry body weight At the end of exposure (post-hatch day 5) the mean fish dry weight from pooled survivors per replicate was determined from the control and of three test groups where no significant mortalities were observed. The fish were dried for at least 24 h at 60 °C. Dry biomass weight was measured to the nearest 0.001 mg.




Physical Properties

Water quality and Temperature, pH value and oxygen saturation were measured
light intensity on each water renewal interval from freshly prepared and old test
measurements 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.
The light intensity on the surface of the test vessels was measured at start of the exposure.




VEHICLE CONTROL PERFORMED: no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

9 d

Dose descriptor:

NOEC

Effect conc.:

200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatch, post hatch survival, length, dry weight

Remarks on result:

other: highest concentration of test item used in test

Effect concentrations 2

Duration:

9 d

Dose descriptor:

LC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: hatch, post hatch survival, length, dry weight

Details on results:

- Mortality/survival at embryo and larval stages:
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 25.0 mg/L; post hatch day 1: 12.5 and 50.0 to 200 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 96 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.


- Overall mortality/survival:
Overall survival at the end of the study was 93 % in the control group and ranged from 83 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). No statistically significant differences were found for the tested concentrations when compared with the control.


- Days to hatch and numbers hatched:
Egg hatch began on study day 3 in the control and all tested concentrations 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 93 %.
Statistical procedures (One Way Analysis of Variance) were applied for study days 3, 4 and 5 (post hatch days -1, 0 and 1). A statistically significant difference was found on post hatch day -1 for the concentration level of 200 mg/L. However, this effect was transient and considered to be biologically not significant.

Swim-up was observed for a 3-day period on study days 5 to 7. Newly hatched fry of the control 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 had swum up. In the test concentrations of 12.5 to 200 mg/L newly hatched fry began to swim up on study day 5 (post hatch day 1). The completion of swim-up in those test concentrations was on study 7 (post hatch day 3). No significance tests were carried out for swim-up.


- Data for length and weight of surviving fish:
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 of 12.5 to 200 mg/L and 50.0 to 200 mg/L, respectively.




- Type of and number with morphological abnormalities:
No biologically significant morphological and behavioural effects were observed in any tested replicate throughout exposure.

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 day 3. Normality test failed for hatch data of study day 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 weight data of study day 9. Normality test failed for post hatch survival data. No transformation was carried out with these data because the data set was not estimated to follow a normal distribution.
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.

Any other information on results incl. tables

Egg Hatch / Hatching Time

Nominal test item concentration [mg/L]	Replicate	Egg hatch [%]
		Post hatch day -2 (Study day 2)	Post hatch day -1 (Study day 3)	Post hatch day 0 (Study day 4)	Post hatch day 1  (Study day 5)	Post hatch day 2   (Study day 6)
200	1	0	30	80	80	80
	2	0	20	100	100	100
	3	0	20	60	80	80
	Mean	0	23   (+)	80	87	87
100	1	0	80	100	100	100
	2	0	70	100	100	100
	3	0	70	90	100	100
	Mean	0	73	97	100	100
50.0	1	0	90	90	100	100
	2	0	80	100	100	100
	3	0	60	70	90	90
	Mean	0	77	87	97	97
25.0	1	0	50	100	100	100
	2	0	80	90	90	90
	3	0	50	100	100	100
	Mean	0	60	97	97	97
12.5	1	0	90	100	100	100
	2	0	70	80	90	90
	3	0	70	100	100	100
	Mean	0	77	93	97	97
Control	1	0	70	90	90	90
	2	0	40	90	90	90
	3	0	70	100	100	100
	Mean	0	60	93	93	93

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

Nominal test item concentration [mg/L]	Study day with maximum hatch	Replicate	Hatched larvae on study day with maximum hatch n/10	Vital Larvae on study day 9 (PHD 5) n/10	Post Hatch survival [%]
200	5 (PHD 1)	1	8	7	88
		2	10	10	100
		3	8	8	100
		Mean	8.7	8.3	96.0
100	5 (PHD 1)	1	10	10	100
		2	10	10	100
		3	10	10	100
		Mean	10.0	10.0	100
50.0	5 (PHD 1)	1	10	10	100
		2	10	10	100
		3	9	9	100
		Mean	9.7	9.7	100
25.0	4 (PHD 0)	1	10	10	100
		2	9	9	100
		3	10	10	100
		Mean	9.7	9.7	100
12.5	5 (PHD 1)	1	10	10	100
		2	9	9	100
		3	10	10	100
		Mean	9.7	9.7	100
Control	4 (PHD 0)	1	9	9	100
		2	9	9	100
		3	10	10	100
		Mean	9.3	9.3	100

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

Nominal test item concentration [mg/L]	Replicate	Live fry on post hatch day 5 (Study day 9) n/10	Overall survival [%]	Mortality [%]
200	1	7	70	30
	2	10	100	0
	3	8	80	20
	Mean	8	83	17
100	1	10	100	0
	2	10	100	0
	3	10	100	0
	Mean	10	100	0
50.0	1	10	100	0
	2	10	100	0
	3	9	90	10
	Mean	10	97	3
25.0	1	10	100	0
	2	9	90	10
	3	10	100	0
	Mean	10	97	3
12.5	1	10	100	0
	2	9	90	10
	3	10	100	0
	Mean	10	97	3
Control	1	9	90	10
	2	9	90	10
	3	10	100	0
	Mean	9	93	7

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

C8/C10-Glucamide AC93/13 caused no significant effects on the short-term toxicity with embryo and sac-fry stages of zebrafish under semi-static conditions up to the dosage level of 200 mg/L (nominal test item concentration). The LC50 on study day 9 (post hatch day 5) was determined > 200 mg/L. The NOEC for the parameters hatch, post hatch and overall survival (mortality), growth (expressed as length and dry weight) was 200 mg/L.. All effect values are given based on the nominal concentrations of the test item C8/C10-Glucamide AC93/13.

Executive summary:

The effects of the test item C8/C10-Glucamide AC93/13 (batch no.:AC93/13) to the embryo and sac-fry stages of fish (Zebrafish /Danio rerio) were determined according to OECD Guideline 212 from 2013‑10‑09 to 2013-10-21, with a definitive exposure phase from 2013-10-09 to 2013-10-18 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 12.5 – 25.0 – 50.0 – 100 – 200 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 ofDanio reriowere exposed per test concentration and control (3 replicates with 10 eggs each), respectively.

 

On day four 93 % 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 LC-values were determined based on the statistical results.

 

The analytical monitoring of the concentrations of C8/C10-Glucamide AC93/13 in the fresh test media (days 0, 5 and 7) and in the corresponding aged test media (days 2, 7 and 9) at all concentration levels and the control was carried out by LC-MS/MS analysis.

The measured recoveries of the test item C8/C10-Glucamide AC93/13 in the freshly prepared test media were in the range of 90 to 111 % of the fraction C8-Glucamide and 92 to 116 % of the fraction C10 -Glucamide of the nominal values, respectively. The measured concentrations of the test item in the corresponding aged test media were in the range of 85 to 115 % of the fraction C8-Glucamide and 86 to 113 % of the fraction C10-Glucamide of the nominal values, respectively.

All effect levels were based on the nominal concentrations of the test item C8/C10-Glucamide AC93/13

NOEC, LOEC: Hatch, Fry Survival and Fry Growth

                     based on nominal test item concentrations [mg/L]

 

Parameter	NOEC	LOEC
Hatch	200	> 200
Post hatch survival	200	> 200
Overall survival	200	> 200
Length	200	> 200
Dry weight	200	> 200

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

                     based on nominal test item concentrations [mg/L]

 

LC50	> 200
95 % confidence interval	Not applicable