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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2017-08-08 to 2017-08-11, with the definitive exposure phase from 2017-08-09 to 2017-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD (2000): Guidance document on aquatic toxicity testing of difficult substances and mixtures. OECD series on testing and assessment no. 23, ENV/JM/MONO(2000)6
Version / remarks:
2000
Deviations:
not applicable
Principles of method if other than guideline:
Principle of guideline test:
An acute immobilization test to Daphnia magna STRAUS was carried out to determine the effects of a Water Accommodated Fraction (WAF) at a loading of 100 mg/L of the test item after 24 and 48 hours of exposure under static conditions in a limit test.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
The water accommodated fraction (WAF) and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start (0h) and at the end (48h) of the exposure.

WAF -- Water accommodated fraction: The aqueous medium containing only the fraction of a muiti-component test item that is dissolved and / or present as a stable dispersion / emulsion under test conditions in the test medium, acc. to OECD Series, No. 23, ENV/JM/MONO(2000)6
Vehicle:
no
Details on test solutions:
Loading level
A WAF with a nominal loading of 100 mg/L was tested in a limit test. The WAF is based on the results of a non-GLP preliminary range finding test conducted at the test facility under static conditions.

For details on the justification of use of WAF refer to 'any other information on materials and methods'.

Preparation of the water accommodated fraction
The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L. An appropriate amount of the test item was weighed out on a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of demineralized water. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and following a separation phase of 1 hour of standing, the homogenous, aqueous phase or WAF was removed by siphoning from the approximate center of the water body. Thereafter, a mineral components of the dilution water were added to the WAF. The resulting water accommodated fraction (WAF) was used in a limit test. The WAF was visually clear throughout the exposure period.

Control
Dilution water without test item incubated under the same conditions as the test groups.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline:
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), Berlin, Germany
- Breeding: Noack Laboratorien GmbH, Sarstedt, Germany
- Feeding during test: no

ACCLIMATION
- Acclimatization period: Acclimatization was not necessary, because the dilution water was equivalent to the culture medium
- Culturing: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 hours illumination, light intensity of max. 1500 lx
- Acclimation conditions (same as test or not): yes
- Type and amount of food: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.
- Health during acclimation: yes
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
approximately 160 to 180 mg CaCO3/L
Test temperature:
19-20.9 °C
pH:
7.38 - 8.28
Dissolved oxygen:
7.73 - 8.98 mg/L
Nominal and measured concentrations:
Nominal conentration: 100 mg/L (loading level of the WAF)
Measured TOC concentration: start: 2.80 mg C/L at, end: 2.58 mg C/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses
- Fill volume: 20 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to OECD 202
- Hardness: 176 CaCl2 x 2 H2O mg/L; original recipe: 293.8 mg/L, modified to achieve a total water hardness of 160 to 180 mg CaCO3/L.
- Culture medium different from test medium: no
- Intervals of water quality measurement:
pH-value / dissolved oxygen concentration: at start and end of the test
Temperature: recorded throughout the period

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16/8 hours Iight/dark cycle
- Light intensity: Diffuse light, Iight intensity of max. 1500 Ix

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility: determined in all groups after 24 and 48 hours

RANGE-FINDING STUDY
- Test concentrations: nominal loading levels of 10 and 100 mg test item/L
- Results used to determine the conditions for the definitive study: yes

TEST PROCEDURE
An acute immobilization test to Daphnia magna STRAUS was carried out to determine the effects of a Water Accommodated Fraction (WAF) at a loading of 100 mg/L of the test item after 24 and 48 hours of exposure under static conditions in a limit test.
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
20 g test solution per replicate were weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by pipette.
Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. No other observations were made.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: effect level of WAF
Duration:
48 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: effect level of WAF
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: effect level of WAF
Duration:
24 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: effect level of WAF
Details on results:
In the WAF with a nominal loading of 100 mg/L of the test item WARADU LG, no effects on Daphnia magna were observed: 48h-EL10 (Daphnia magna, immobility) > 100 mg/L (nominal), 48h-EL50 (Daphnia magna, immobility) > 100 mg/L (nominal).

Please find a summary table of results in 'any other information on results'.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: reported effect levels refer to the WAF
Results with reference substance (positive control):
EC50-Value (with 95% confidents limits) of the reference item potassium dichromate based on nominal concentrations [mg/L], (0 - 24 hours):
EC50 = 1.90 mg/L, 95% confidence limits: 1.43 - 2.69 mg/L

Validity range:
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
Reported statistics and error estimates:
EC-values and statistical analyses for reference item
EC50-value was calculated for the reference item by linear dose response regression. The respective 95 % confidence limits for the
EC50 were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if
manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism5, GraphPad Software, Inc.
- Excel, Microsoft Corporation

Immobilization Rates after 24 and 48 hours of Exposure during the Definitive Test

WARADUR® LG
Nominal loading level in the WAF
[mg/L]
 % immobilisation
24 hours 48 hours
Replicates Replicates
1 2 3 4 Mean 1 2 3 4 Mean
100 0 0 0 0 0 0 0 0 0 0
Control 0 0 0 0 0 0 0 0 0 0

Absolute Numbers of immobile Daphnids after 24 and 48 h of Exposure in the Definitive Test

WARADUR® LG
Nominal loading level in the WAF
[mg/L]
24 hours 48 hours
Replicates Replicates
1 2 3 4 Mean 1 2 3 4 Mean
100 0/5 0 / 5 0/5 0 / 5 0/20 0/5 0 / 5 0/5 0 / 5 0/20
Control 0/5 0 / 5 0/5 0 / 5 0/20 0/5 0 / 5 0/5 0 / 5 0/20

Measured Total Organic Carbon (TOC) Concentrations during the Definitive Test

Measured TOC concentration* [mg/L]
WARADUR® LG
Nominal loading level of the test item in WAF
[mg/L]

Start of the exposure,

0 hours

End of the exposure,

48 hours

100 2.8 2.58
Control < LOQ < LOQ

* = measured TOC concentration, mean value of 2 injections

Validity criteria fulfilled:
yes
Conclusions:
In the WAF with a nominal loading of 100 mg/L of the test item WARADU® LG, no effects on Daphnia magna were observed: 48h-EL10 (Daphnia magna, immobility) > 100 mg/L (nominal), 48h-EL50 (Daphnia magna, immobility) > 100 mg/L (nominal).
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item WARADUR® LG were determined according to OECD 202 (2004) and in compliance with GLP.

The study was conducted under static conditions over a period of 48 hours with one Water Accommodated Fraction (WAF) of the test item prepared at a loading level of 100 mg/L. The 100 mg/L WAF was visually clear throughout the exposure period. The loading level of the test item WARADUR® LG and the control was analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (0 hours) and at the end of the exposure (48 hours). Measured TOC concentrations were similar at the start and end of the test, demonstrating a stable WAF of the test item during exposure.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control. Per definition of the WAF, all terms related to concentration levels were given as loading levels, because partly dissolved compounds and mixtures cannot be related to concentrations. Therefore, the EL10 and EL50 were based on the nominal loading level. No immobility was observed at the limit loading level and in the control after 24 and 48 hours. Other effects were not observed. Therefore, the derived effect concentrations were: 48h-EL10 > 100 mg/L (nominal) and 48h-EL50 > 100 mg/L (nominal).

The validity criteria of the test guideline were fulfilled and the study was considered adequate and reliable for the environmental hazard assessment.

Description of key information

In five reliable studies (reliability category 1) with different members of the Montan waxes category no biologically relevant toxicity to Daphnia magna was observed using water accommodated fractions with nominal loading rates up to the limit concentration of 100 mg/L recommended by the OECD 202 guideline.

EC50/LC50 for freshwater invertebrates > water solubility limit (OECD 202, 2002 / 2017)

Key value for chemical safety assessment

Additional information

Please refer to CSR for further details on study summaries of different members of the Montan waxes category as well as Read-across justification in section 13 'assessment reports'.

Five reliable (reliability category 1) studies on the acute toxicity towards invertebrates (according to OECD 202, compliant to GLP) for Montan waxes, types LG, NaV 101, WE 4, CaV 102 and E are available, meeting the validity criteria of the OECD-guideline. Due to the poor water solubility of Montan waxes, water accommodated fractions (i.e. saturated solutions) were tested, prepared separately at each nominal concentration used in the respective studies. All (no)effect concentrations refer to the nominal loading levels used to prepare the saturated solutions. A static test design was applied.

For Montan waxes, types LG, WE 4, and E consistently no toxicity to Daphnia magna could be found in these tests. Water accommodated fractions prepared at nominal concentrations far above the water solubility limit of the waxes and even above to nominal limit concentration recommended by the OECD 202 guideline (100 mg/L) were tested.

For Montan waxes, type NaV 101 upon testing of water accommodated fractions prepared at very high loading rates (500 mg/L and higher) immobilisations occurred. Based on these results an EC50 (48 h) and EC10 (48 h) of 440 and 320 mg/L, respectively, were derived. However, consistently no immobilized daphnids were observed for all other concentration groups, i.e. 50, 100 and 250 mg/L. Experimentally determined water solubility for Montan waxes, type NAV 101 is only 20 mg/L and the OECD 202 guideline recommends a limit concentration of 100 mg/L, which is already far above the solubility limit. Effect concentrations determined based on nominal test item concentrations even far above the limit concentration of 100 mg/L as stated by the OECD guideline are regarded as not scientifically valid: Minor components of higher solubility present in the complex substance possibly responsible for toxicity are given overly high weight due to the extraordinary high nominal concentrations used to prepare WAF. Therefore, study results are interpreted thus that Montan waxes, type NaV 101 produced no immobilizations up to the nominal limit concentration of 100 mg/L: EC0 (48 h; immobilization) ≥ 100 mg/L (nominal, WAF).

Similarly, for Montan waxes, type CaV 102 water accommodated fractions of very high concentrations (up to 10 g/L) far above water solubility (27 mg/L experimentally determined water solubility) had been tested. Up to 100 mg/L no more than 10% immobilization was observed, at the highest concentration of 10 g/L not more than 30% of daphnids were immobilized and an EC50 could not be determined. A validity criterion of OECD 202 guideline states that the Acute Immobilisation Test with Daphnia magna is valid, if no more than 10 per cent of daphnids are immobilized in the control. Thus given the inherent test design, biologically relevant effects must be above 10 per cent. Thus, taking this into account and including the arguments stated for Montan waxes, type NaV 101 in the preceding paragraph, up to the nominal limit concentration of 100 mg/L (water accommodated fractions) no biologically relevant effects were observed for Montan waxes, type CaV 102.

In conclusion, four reliable studies (reliability category 1) on acute immobilisation of Daphnia magna performed with different members of the Montan waxes category are available. Consistently no biologically relevant toxicity to Daphnia magna was observed using water accommodated fractions with nominal loading rates up to the limit concentration of 100 mg/L recommended by the OECD 202 guideline. Therefore, members of the category of Montan waxes are concluded to be acutely nontoxic to invertebrates.