cyclic C12/14-Glucamide;cyclic N-methyl-N-dodecanoyl/tetradecanoyl-glucamine;anhydro-N-dodecanoyl/tetradecanoyl-N-methylglucamine;Amides, C12-C14, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated

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:

from 2021-04-12 to 2021-04-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item cyclic Glucamide C12-14
Batch number S148866
Purity 100 % (UVCB)
Sum formula main compounds : C19H37NO5 (C12-acyl-derivative);
C21H41NO5 (C14-acyl-derivative)
Molecular weight main compounds : 359.5 g/mol (C12-derivative); 387.5 g/mol (C14-derivative)
Density 1.07 g/cm3
Water solubility soluble/miscible
Appearance slightly turbid brown solid mass
Expiry date 2022-09-30
Recommended storage Room temperature, dry

Analytical monitoring:

yes

Details on sampling:

Chemical Analysis of Test Item Concentrations

Determination of the test item
Samples were analyzed with a LC-MS/MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated according to Annex I.

Sampling schedule
The limit loading rate and the control were analytically verified in in the fresh media at the start of exposure and at the renewal of the test solutions (0 and 72 hours) as well as in the 24-hours old media at the renewal and at the end of the exposure (24 and 96 hours).

Vehicle:

no

Details on test solutions:

Test method A semi-static test with daily renewal of the test media was performed.

Limit loading
A water accommodated fraction (WAF) of 4.00 mg/L was tested as a threshold concentration in a limit test. The selection of the test concentration is based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia toxicity test (EL50 (48 h) = 3.68 (95% CI: 3.11 – 4.29) mg/L, nominal test item loading) and of an alga toxicity test (ErL50 (0-72 h) = 11.8 (95% CI: 11.0 – 13.8) mg/L, nominal test item loading) and (EYL50 (0-72 h) = 5.32 (95% CI: 4.88 – 5.81) mg/L, nominal test item loading).

Water Accommodated Fraction
A water accommodated fractions (WAF) was prepared because the test item an UVCB substance with constituents of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2018).
Using this approach, aqueous media was prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.


Preparation of the water accommodated fractions
One water accommodated fraction (WAF) was prepared with a nominal loading rate of the test item of 4.00 mg/L.
For the loading rate, an appropriate amount of the test item was weighed out and transferred into a measuring flask with an appropriate amount of dilution water. The WAF’s were treated with ultrasound for 5 minutes at room temperature. This procedure was carried out for each study day.

Control
7 fish in dilution water (without test item but treated in the same way as the test solution) 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
Danio rerio (zebrafish)
Vertebrata, Gnathostomata, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae

Reason for the selection of the test system
According to the guideline, Danio rerio is suitable for this kind of study.

Origin
All fish used in the test were gained at Noack Laboratorien GmbH from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)

Holding
Holding was performed at the test facility at 23 ± 2 °C and diffuse light (540 – 1000 Lux, photoperiod of 12 to 16 h light / 12 to 8 h dark). The water was changed at least once per week. The dissolved oxygen concentration was more than 80% of the air saturation value.
Zebrafish with at least 9 days of acclimatization and mortality < 5% within these days prior to the start of the exposure were used in the test.
No mortality was observed during this time. No disease treatments were administered throughout holding and testing.

Water 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: 40 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Oxygen saturation: ≥ 80 % of air saturation value
Acidity: 0.1 mmol/L (recent measurement 2021-01-04)
Alkalinity: 0.6 mmol/L (recent measurement 2021-01-04)
Conductivity: 150 µS/cm (recent measurement 2021-01-04)

Acclimatization
A sufficient number of fish were acclimatized to dilution water and environmental conditions equivalent to the test conditions for at least 9 days.

Feeding
Food was provided 3 times per week. Food was given to satiation (4% of the fish body weight) per feeding day. The test fish were not fed 24 h before test start.

Food Sera Vipan; SERA GMBH, 52518 Heinsberg, Germany

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

TOC and Total Hardness from the Dilution Water

Parameter Study Days [mg/L]
TOC 0 to 4 < 2
Total Hardness 0 to 4 67

Test temperature:

Water Temperature in the Control (Continuous Measuring) in the Definitive Test

Period of Measurements 2021-04-12 to 2021-04-16
Minimum Temperature [°C] 22.8
Maximum Temperature [°C] 24.2
Mean Temperature [°C] 23.4

pH:

see " Any other information on materials and methods incl. tables"

Dissolved oxygen:

see " Any other information on materials and methods incl. tables"

Nominal and measured concentrations:

A water accommodated fraction (WAF) of 4.00 mg/L was tested as a threshold concentration in a limit test. The selection of the test concentration is based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia toxicity test (EL50 (48 h) = 3.68 (95% CI: 3.11 – 4.29) mg/L, nominal test item loading) and of an alga toxicity test (ErL50 (0-72 h) = 11.8 (95% CI: 11.0 – 13.8) mg/L, nominal test item loading) and (EYL50 (0-72 h) = 5.32 (95% CI: 4.88 – 5.81) mg/L, nominal test item loading).

The measured concentrations of the test item in the fresh and the old media were between 80 and 120 % throughout the test.

Details on test conditions:

Duration of the test 96 hours

Test vessel Glass aquaria of 3 L were used (dimensions: 11.5/13/20 cm, depth of water: approx. 17 cm) and covered with a glass plates.

Test volume About 2.5 L per vessel

Aeration No aeration was provided.

Dilution water Same as used for holding

Number of fish 7 zebrafish were used for the limit concentration and the control. For the whole study 14 fish were used.

Loading, length of fish Fish density in the tanks was 0.132 g fish per liter test solution. Average body length at the test end: 1.46 cm
Average body weight at the test end: 0.047 g

Age of fish Juvenile

Replicates One replicate each for the loading and the control

Introduction of fish The fish were introduced randomly to individual replicates.

Feeding of test fish The fish were not fed during the exposure.

Water temperature (target) 21 - 25 °C, controlled at ± 1 °C

 
Dissolved oxygen Not less than 60% of air saturation value.
Concentration (target)

Light intensity (target) 540 – 1000 Lux

Photoperiod A photoperiod (light / dark cycle: 16 / 8 h) was maintained during exposure.

Observation intervals Observations were made after 2 ± 0.5 h and 5 ± 1 h after the start of the exposure. On days 1-4 of the test, all vessels with living fish were inspected twice per day.

Criteria of effects 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 behavior, respiratory function, pigmentation, etc.).

Measurement of fish size and weight
For measurements of size and weight all control fish were used at the end of the exposure.


Water quality and light intensity measurements
The pH-value, temperature and oxygen saturation were measured daily in all test vessels at the start and the end of exposure intervals from fresh and old test media. Chlorine and nitrate were measured from each batch of dilution water used in the test.
Total hardness and TOC were determined at the start of the exposure in the dilution water. 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 the start of the exposure.
The solution appearance and behavior was observed daily before and after renewal.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

>= 4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Biological Data

Observations and effects on behavior in the test vessels are given in the table below. No mortality, no presence of visible abnormalities in regard to equilibrium, appearance, swimming behavior, ventilatory behaviour function and no additional clinical signs were observed. All effect levels are given based on the nominal test item loading. The LL0 after 96 hours was the nominal test item loading rate of ≥ 4.00 mg/L.


Observations of Sub Lethal Effects and Normal Behavior in the Test Vessels
(n = 7, number of fish)

Nominal Test Item loading
[mg/L] Effect *


Number of fish affected at observation time [hours]
day 0 day 1 day 2 day 3 day 4
2 4 23 30 47 53.5 72 78 94 96
4.00 (1) 7 7 7 7 7 7 7 7 7 7
Control (1) 7 7 7 7 7 7 7 7 7 7
*) The numbers in brackets correspond to the following observations:
(1) = Normal behavior

Cumulative Mortality % in the Test Vessels
Nominal Test Item loading
[mg/L] Cumulative mortality [%] at observation time [hours]
day 0 day 1 day 2 day 3 day 4
2 4 23 30 47 53.5 72 78 94 96
4.00 0 0 0 0 0 0 0 0 0 0
Control 0 0 0 0 0 0 0 0 0 0

The environmental conditions (pH-value, temperature and oxygen-saturation) were determined to be within the acceptable limits. Light intensity was measured at the start of the exposure and ranged from 693 to 773 (mean value 735) Lux.

Sublethal observations / clinical signs:

Measured Exposure Concentrations during the Definitive Test

The concentration of the test item cyclic Glucamide C12-14 was analytically verified via LC-MS/MS in fresh media at the start of the exposure (0 hours and 72 hours) and in the corresponding old media at the end of the exposure (24 hours and 96 hours) in the limit test item concentration and the control.

Measured Concentrations of the active substances of the Test Item cyclic Glucamide C12 14 during the Definitive Test

Sampling date:       0 hours,

Fresh medium       24 hours,

Old medium       72 hours,

Fresh medium       96 hours,

Old medium

Nominal

concentration of       C12-acyl-derivative

the test item [mg/L]       the active substance

[mg a.s./L]       Meas. conc.

[mg a.s./L]       %       Meas. conc.

[mg a.s./L]       %       Meas. conc.

[mg a.s./L]       %       Meas. conc.

[mg a.s./L]       %

4.00       2.64       2.46       93       2.78       105       2.80       106       2.63       99

Control       < LOQ       < LOQ       < LOQ       < LOQ

Sampling date:       0 hours,

Fresh medium       24 hours,

Old medium       72 hours,

Fresh medium       96 hours,

Old medium

Nominal

concentration of       C14-acyl-derivative

the test item [mg/L]       the active substance

[mg a.s./L]       Meas. conc.

[mg a.s./L]       %       Meas. conc.

[mg a.s./L]       %       Meas. conc.

[mg a.s./L]       %       Meas. conc.

[mg a.s./L]       %

4.00       0.792       0.800       101       0.692       87       0.913       115       0.730       92

Control       < LOQ       < LOQ       < LOQ       < LOQ

Meas. conc.       = measured concentration of the active substance, dilution factor taken into account

%       = percent of the nominal concentration of the test item

a.s.       = active substance

LOQ       = limit of quantification of the analytical method (0.500 mg test item/L, corresponding to

0.331 mg C12 acyl-derivative and 0.0990 C14-acyl-derivative)

Validity criteria fulfilled:

yes

Conclusions:

The effect level is given based on the nominal test item loading of cyclic Glucamide C12-14. The threshold concentration of the test item at the nominal loading of 4.00 mg/L was found to have neither lethal nor sub lethal effects on zebrafish.
In conclusion, the LL0 corresponds to the nominal loading of ≥ 4.00 mg test item/L.

Executive summary:

The acute toxicity of the test item cyclic Glucamide C12-14 (batch no. S148866) to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (2019) from 2021-04-12 to 2021-04-16 at the test facility.

An acute toxicity test under semi-static conditions was performed. The test item cyclic Glucamide C12-14 is a slightly turbid brown solid mass. The test item is soluble/miscible in water.

A water accommodated fraction (WAF) as a limit loading of 4.00 mg/L was tested as a threshold concentration. The concentration was based on the derivation of a threshold concentration (TC) from the data of the test item of results of a daphnia toxicity test (EL50 (48 h) = 3.68 (95% CI: 3.11 – 4.29) mg/L, nominal test item loading) and of an alga toxicity test (ErL50 (0-72 h) = 11.8 (95% CI: 11.0 – 13.8) mg/L, nominal test item loading) and (EYL50 (0-72 h) = 5.32 (95% CI: 4.88 – 5.81) mg/L, nominal test item loading).

The study was performed over a period of 96 hours under semi-static conditions with daily renewal of the test solutions. Seven test organisms were exposed to the limit concentration and the control. No mortality was observed in the definitive test. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

The limit loading of the test item cyclic Glucamide C12-14 and the control was analytically verified via LC-MS/MS in fresh media at the start of the exposure (0 hours and 72 hours) and in the corresponding old media at the end of the exposure (24 hours and 96 hours).

The measured concentrations of the test item in the fresh and the old media were between 80 and 120 % throughout the test.

All effect values given were based on the nominal loading rate of the test item.

All validity criteria of the OECD 203 (2019) test guideline were met. The results of the study are summarized in Table 1. The threshold concentration of the test item at the nominal loading rate of 4.00 mg/L was found to have neither lethal nor non lethal effects on zebrafish.

LL-Values (96 hours) of the Test item

based on the nominal test item loading rate [mg/L]

LL0 =

Highest test item loading with

0% mortality after 96 h       ≥ 4.00 mg/L (nominal loading)

Description of key information

The effect level is given based on the nominal test item loading of cyclic Glucamide C12-14. The threshold concentration of the test item at the nominal loading of 4.00 mg/L was found to have neither lethal nor sub lethal effects on zebrafish.
In conclusion, the LL0 corresponds to the nominal loading of ≥ 4.00 mg test item/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Remarks:

LL50 LL0 ≥ 4 mg/L

Effect concentration:

> 4 mg/L