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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:
2014-06-03 to 2014-07-24
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Five water accommodated fractions (WAF) were prepared with nominal loading levels of the test item of 12.5 – 25.0 – 50.0 – 100 - 200 mg/L.
- Sampling method: The content of free silicon was analytically verified by spectrophotometry with a validated cuvette test at the start (0 h) and at the end of the exposure (48 h) in all loading levels (pH-neutralised and un-neutralised) and in the control as specified below.
At the start of the exposure, sampling was carried out after preparation of the WAFs.
At the end of the exposure (48 h), samples were taken from additional replicates with test media, but without daphnids, which were incubated under test conditions until sampling.
- Sample storage conditions before analysis: All original samples were stored in the refrigerator until sample preparation. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For each loading level, an appropriate amount of the test item was weighed out and transferred into a brown glass flask with an appropriate amount of dilution water. This dispersion was shaken for 24 h with 20 rpm at room temperature. After a separation phase of 1 h, the aqueous phase or WAF was removed by siphoning from the approximate centre of the flask to give the WAF with a definitive loading level.
- Eluate: Dilution water
- Differential loading: 12.5 – 25.0 – 50.0 – 100 - 200 mg/L.
- Controls: Dilution water without test item under the same test conditions as the test groups
- pH-Controls: The WAFs with a loading level of 100 and 200 mg/L were adjusted to a pH in the range of 7.0 ± 0.2 by addition of 1 M HCl and tested under the same conditions as the un-neutralised loading levels
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain: Clone 5
- Source:
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Age at study initiation (mean and range, SD): 2 to 24 h old daphnids from a healthy stock were used for the study
- Method of breeding:
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +- 2°C, in an incubator, 16 h illumination; light intensity
of max. 20 µE x m^-2 x s^-1
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Cultur feeding: 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 > 10^6 cells/mL
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: At least 2 h in dilution water.
- Acclimation conditions (same as test or not): Same as test conditions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
256 mg CaCO3/L
Test temperature:
18 - 22°C, constant within ± 1°C
pH:
pH at the start of the exposure (0 h)
(measured in one additional replicate per loading level (pH-neutralised and un-neutralised) and control

Nominal
loading level of the
water accommodated fraction
[mg/L] pH-value
200*
(pH-neutralised) 6.87
100*
(pH-neutralised) 6.88
200 11.39
100 10.84
50.0 7.78
25.0 7.54
12.5 7.92
Control 7.64

pH at the end of the exposure (48 h)
(measured in all replicates)

Nominal
loading level of the pH-value
water accommodated fraction Replicates
[mg/L]
1 2 3 4
200*
(pH-neutralised) 7.15 7.14 7.15 7.15
100*
(pH-neutralised) 7.32 7.21 7.20 7.21
200 7.68 7.70 7.71 7.73
100 7.59 7.60 7.60 7.61
50.0 7.66 7.64 7.61 7.59
25.0 7.70 7.70 7.69 7.70
12.5 7.56 7.59 7.63 7.63
Control 7.29 7.37 7.40 7.41

* = WAF neutralised by addition of 1 M HCl to a pH in the range of 7 ± 0.2.
Dissolved oxygen:
Dissolved O2-concentration at the start of the exposure (0 h)
(measured in one additional replicate per loading level (pH-neutralised and un-neutralised) and control

Nominal
loading level of the
water accommodated fraction Dissolved O2-concentration
[mg/L] [mg/L]
200*
(pH-neutralised) 8.80
100*
(pH-neutralised) 8.83
200 8.84
100 8.79
50.0 8.68
25.0 8.67
12.5 8.94
Control 9.06

Dissolved O2-concemtration at the end of the exposure (48 h)
(measured in all replicates)

Nominal Dissolved O2-concentration
loading level of the [mg/L]
water accommodated fraction Replicates
[mg/L]
1 2 3 4
200*
(pH-neutralised) 8.66 8.64 8.68 8.68
100*
(pH-neutralised) 8.55 8.60 8.53 8.58
200 8.72 8.64 8.74 8.75
100 8.86 8.89 8.88 8.71
50.0 8.86 8.82 8.84 8.86
25.0 8.73 8.80 8.83 8.83
12.5 8.75 8.77 8.83 8.83
Control 8.80 8.77 8.69 8.69

* = WAF neutralised by addition of 1 M HCl to a pH in the range of 7 ± 0.2.
Nominal and measured concentrations:
See window "Any other information on materials and methods incl. tables"
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Aeration: None
- 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: ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123

- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure (0 h):
pH-value DissolvedO2-concentration[mg/L] Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
7.64 9.06 21.2 680 256

- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: test start (0h) and test end (48h)

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 20 µE x m^-2 x s^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobilisation, 24 h interval

TEST CONCENTRATIONS
- Test concentration: 12.5 – 25.0 – 50.0 – 100 - 200 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: not applicable
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: not applicable
Duration:
24 h
Dose descriptor:
EL100
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: not applicable
Duration:
48 h
Dose descriptor:
EL100
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: not applicable
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: not applicable
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 123 to >200
Duration:
24 h
Dose descriptor:
EL10
Effect conc.:
105 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 98.1 to 114
Duration:
48 h
Dose descriptor:
EL10
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 80.7 to 132
Details on results:
See below
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 h. The EC50-value with 95 % confidence interval (CI) was calculated by sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.82 mg/L (CI 1.66 - 2.15 mg/L)
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

The percentage immobility, determined in all test item loading levels (pH-neutralised and un-neutralised) and the control after 24 and 48 h under static conditions, is presented in the table below.

 

Immobilisation Rate safter 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Celitement

IMMOBILISATION [%]

Nominal

loading level of the

water accommodated fraction

[mg/L]

24 h

48 h

Replicates

Replicates

1.

2.

3.

4.

MV

1.

2.

3.

4.

MV

200**

(pH-neutralised)

 0

 0

 0

 0

 0

 0

 0

 0

 0

 0

100*

(pH-neutralised)

 0

 0

20

 0

 5

 0

 0

20

 0

 5

200

20

20

 0

40

20

20

60

40

80

50

100

 0

 0

 0

20

 5

 0

 0

 0

40

10

 50.0

 0

 0

 0

 0

 0

 0

 0

 0

 0

 0

  25.0

 0

 0

 0

 0

 0

 0

 0

 0

 0

 0

 12.5

 0

 0

 0

 0

 0

 0

 0

 0

 0

 0

Control

 0

 0

 0

 0

 0

 0

 0

 0

 0

 0

MV   = mean value

*       = The pH of the concentration level of 100 mg/L was adjusted with 1 M HCl from pH 10.84 to pH 6.88.

**      = The pH of the concentration level of 200 mg/L was adjusted with 1 M HCl from pH 11.39 to pH 6.87.

All test item loadings (pH-neutralised and un-neutralised) and the controlwere clear throughout exposure.

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal concentrations of the test item Celitement, the 48 h-EC50 for Daphnia magna was 200 mg/L (95 % confidence limits: 123 to > 200 mg/L). The NOEC after 48 h was 100 mg/L. The LOEC after 48 h was 200 mg/L.
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item Celitement (batch number: Not specified) were determined according to OECD 202 (2004) from 2014-06-03 to 2014-07-24, with the definitive exposure phase from 2014-07-22 to 2014-07-24, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The test item is a UVC substance of amorphous and crystalline constituents, solid and poorly soluble in water (hydration reaction with water). In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF)* of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents.Using this approach, aqueous media were prepared by shaking the test item with dilution water for 24 hours to ensure equilibration between the test item and the water phase.At the completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.
Based on a non GLP preliminary range finding test, the definitive Acute Immobilisation Test to Daphnia magna was performed with 5 WAFs of the test item Celitement with nominal loading levels in the range of 12.5 to 200 mg/L, prepared in a geometric series with a separation factor of 2. The WAFs with a nominal loading level with 100 and 200 mg/L were adjusted to a pH in the range of 7.0±0.2 by addition of 1 M HCl and tested under the same conditions as the un-neutralised loading levels. Twenty daphnids were exposed to each loading level, the pH-neutralised loading levels and the control.

The content of free silicon in the tested un-neutralised and pH-neutralised WAFs of the test item Celitement and the control was analytically verified by spectrophotometric analysis at the start of the exposure (0 h) and at the end of the exposure (48 h).
The measured content of free silicon in the test item loadings (pH-neutralised and un-neutralised) at the end of the exposure (48 h) were in the range of 85 to 101 % of the initially measured values (at 0 h).

The recoveries of free silicon at the end of the exposure (48 h) were within ±20 % of the initially measured values at the start of the exposure (at 0 h). This indicates that the test item was successfully maintained for the duration of the test. The effect levels given are based on the nominal loading levels of the test item Celitement.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

*) Water accommodated fraction (WAF): The aqueous medium containing only the fraction of a multi-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.

EL10-, EL50- (with 95 % Confidence Limits), EL100-Values, NOEL and LOEL

(based on the nominal loading levels of the test item Celitement)

Endpoints

 

 

Test

duration

[h]

Celitement

Nominal

loading level

[mg/L]

95 % confidence limits

[mg/L]

EL10

24

  105

98.1 – 114

48

  100

80.7 – 132

EL50

24

> 200

Not applicable

48

  200

123 – > 200

EL100

24

> 200

Not applicable

48

> 200

Not applicable

NOEL

48

  100

Not applicable

LOEL

48

  200

Not applicable

Description of key information

No acute (short term) toxicity to aquatic invertebrates due to a Klimisch 1 study result.

Key value for chemical safety assessment

Additional information

Short-term toxicity testing with Daphnia magna was carried out according to the OECD 202 guidance taking into account GLP and thus resulting in a Klimish 1 score. Based on the nominal loading levels of Celitement the 48-h EL10 for Daphnia magna was estimated to be 100 mg/L. The NOEL after 48 h was determined to be 100.0 as the highest tested loading level with no biologically significant effect (Immobilization rates of < 10 % are seen to be not significant according to the guideline). The LOEL after 48 h was determined to be 200 mg/L as the lowest tested loading level with a biologically significant effect (Immobilization rates of > 10 % are seen to be significant according to the guideline)