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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:
2015-03-16 to 2015-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Test Concentrations/Loadings :The saturated solution and further four dilution levels prepared out of
the saturated solution with dilution water in a geometrical series with a separation factor of 2, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 %.

This corresponds to the nominal concentrations of:
0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L.


The loading levels are based on a proposal of the sponsor.


-Sampling method: The test concentrations of Disperse Blue ANT were analytically
verified via HPLC-UV in the saturated solution and the control at the start of the exposure (0 hours).
At the start of the exposure (0 hours), sampling was carried out after preparation of the loading levels.
As the measured test item concentration was below the LOQ in the saturated solution, the test item concentrations at the other loading levels were considered to be also below the LOQ. At the end of the exposure (48 hours), no analytical monitoring was carried out, because a measured test item concentration above the LOQ was not observed at the start of the exposure.


- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample 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: The saturated solution (5 mg/L test item were weighed out) was prepared with dilution water one day before the start of the exposure
(at -24 hours).

The test item was dispersed in the dilution water by stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature.
After completion of stirring, the test item dispersion was filtered with a membrane filter (0.2 µm, RC, MACHEREY-NAGEL) to achieve the saturated
solution


- Eluate: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, was used.
- Differential loading: The saturated solution and further four dilution levels prepared out of the saturated solution with dilution water in a
geometrical series with a separation factor of 2, were tested as follows:
6.25 - 12.5 - 25.0 - 50.0 - 100 %.
This corresponds to the nominal concentrations of:
0.313 - 0.625 - 1.25 - 2.50 - 5.00 mg/L.



- Controls: Dilution water without test item tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No
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): Less than 24 h old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels 24 h before the start of the exposure and discarded. The juveniles born within the 24 h preceding the exposure were used for the test. No first brood progeny was used for the test. Daphnids were not used for the test if the culture contains ephippia, adults do not
produce young before day 12, more than 20 % of the culture stock die in the two days preceding the test, adults in the culture do not produce an
average of three young per adult per day over the 7-day period prior to the test and if they have been used in any portion of a previous test.
- 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
Culture feeding: The culture daphnids were 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 were cultured at the test facility.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: Not necessary
- Acclimation conditions (same as test or not): Same as test conditions.
- Health during acclimation (any mortality observed): No mortality observed.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
0 h: 166 mgCaCO3/L
Test temperature:
20 ± 2°C
20.1 °C
pH:
pH at the Start of the Exposure (0 h)
(measured in one additional replicate per loading level and control)

Nominal
loading rate
of the test item pH-value
[%] [mg/L]
100 5.00 7.65
50.0 2.50 7.63
25.0 1.25 7.64
12.5 0.625 7.65
6.25 0.313 7.60
Control 7.42



Nominal
loading rate
of the test item pH-values

Replicates
[%] [mg/L] 1 2 3 4
100 5.00 7.37 7.38 7.40 7.42
50.0 2.50 7.33 7.34 7.34 7.32
25.0 1.25 7.34 7.35 7.36 7.31
12.5 0.625 7.36 7.24 7.26 7.29
6.25 0.313 7.28 7.26 7.28 7.34
Control 7.34 7.34 7.34 7.34

Dissolved oxygen:
Dissolved O2-concentration at Start of the Exposure (0 h)
(measured in one additional replicate per loading level and control)

Nominal
loading rate
of the test item Dissolved O2 concentration
[%] [mg/L] [mg/L] [% saturation]
100 5.00 5.74 65.2
50.0 2.50 8.15 92.9
25.0 1.25 8.38 95.3
12.5 0.625 8.31 94.5
6.25 0.313 7.87 89.8
Control 7.42 97.2



Dissolved O2-concentration at the End of the Exposure (48 h)
(measured in all replicates)

Nominal loading rate Dissolved O2 concentration
of the test item
[%] [mg/L] [mg/L] [%]
Replicates Replicates
1 2 3 4 1 2 3 4
100 5.00 7.73 7.48 7.62 7.71 87.7 85.0 86.6 87.7
50.0 2.50 7.39 7.51 6.88 6.52 83.6 85.1 78.0 73.9
25.0 1.25 7.71 7.72 7.73 6.23 87.1 87.1 87.2 70.4
12.5 0.625 7.82 5.72 5.75 6.59 88.6 64.6 64.8 74.5
0.625 0.313 5.31 6.01 6.31 7.76 60.7 68.3 71.6 88.3
Control 8.03 8.10 8.02 6.75 89.5 90.0 89.6 75.0

Nominal and measured concentrations:
The concentrations of the test item Disperse Blue ANT in the saturated solution and in the control were analytically verified via HPLC-UV at the start of the exposure (0 hours).

The measured concentrations of the test item at the start of the exposure (0 hours) were below the LOQ of 0.5 mg/L of the test item in the saturated solution and in the control. Concentrations of the test item in all tested loading levels were considered to be all below the LOQ throughout the exposure. Due to this fact, analytical monitoring of the other dilution levels at the start of the exposure and the analytical monitoring of all loading levels at the end of the exposure was
not carried out in agreement with the sponsor.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (5.5 (ID) x 9 (H) cm), 125 mL capacity, loosely covered with watch glasses
- Test volume: 100 mL (water depth: approximately 5 cm)
- 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: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
Composition of the Culture Medium according to ELENDT (1990)

Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2  0.8


- Water Quality Parameters of the Dilution Water prior to the Start of the Exposure Intervals (0 and 24 h):
pH-value DissolvedO2-concentration Temperature[°C] Conductivity[µS/cm] Total hardness [mg CaCO3/L]
[mg/L] [% saturation]
0 h: 7.42 8.67 97.2 21.0 465 166

- Culture medium different from test medium: No
- Intervals of water quality measurement: Static test

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

RANGE FINDING STUDY
None


TEST CONCENTRATIONS
- Test concentrations: The saturated solution and further four dilution levels prepared out of the saturated solution with dilution water in a geometrical series with a separation factor of 2, were tested as follows: 6.25 - 12.5 - 25.0 - 50.0 - 100 %.
This corresponds to nominal loading levels of: 0.313 – 0.625 – 1.25 – 2.50 – 5.00 mg/L.
The loading levels are based on a proposal of the sponsor.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
See below
Results with reference substance (positive control):
The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (CI) was calculated by
sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50: 1.51 mg/L (CI 1.08 - 2.10 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours 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.


Reported statistics and error estimates:
The EL-values after 24 and 48 hours and the NOEL and LOEL after 48 hours were estimated directly from the observation data, without mathematical
calculation.

An EC50-value was calculated only for the reference item by sigmoidal dose-response regression. The 95 % confidence limits for the EC50 were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

The percentage immobility, determined in all test and control groups after 24 and 48 hours, is given in the table below. The study was performed under static conditions.

 

Immobilisation Rates after 24 and 48 hours of Exposure in the Definitive Test

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

Disperse Blue ANT

IMMOBILISATION [%]

Nominal

loading rate

of the test item

24 hours

48 hours

Replicates

Replicates

[%]

[mg/L]

1

2

3

4

MV

1

2

3

4

MV

100*

5.00*

0

0

0

0

0

0

0

0

0

0

 50.0

2.50

0

0

0

0

0

0

0

0

0

0

 25.0

1.25

0

0

0

0

0

0

0

0

0

0

 12.5

0.625

0

0

0

0

0

0

0

0

0

0

   6.25

0.313

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

 

At the saturated solution with a nominal loading of 5.00 mg/L of the test item Disperse Blue ANT and at any of the tested dilution levels, no significant effects were observed onDaphnia magna.

 

According to OECD Guidance Document No 23 (2000) if an effect cannot be detected in a test with a saturated solution the result has to be reported as no toxic effects at saturation. Therefore,exposures are 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 saturated solution.

The tested solutions were visually clear throughout the exposure period.

The concentrations of the test item Disperse Blue ANT in the saturated solution and in the control were analytically verified via HPLC-UV at the start of the exposure (0 hours).  

The measured concentrations of the test item at the start of the exposure (0 hours) were below the LOQ of 0.5 mg/L of the test item in the saturated solution and in the control. Concentrations of the test item in all tested loading levels were considered to be all below the LOQ throughout the exposure. Due to this fact, analytical monitoring of the other dilution levels at the start of the exposure and the analytical monitoring of all loading levels at the end of the exposure was not carried out in agreement with the sponsor.The analytical results are presented in the table below.

 

 Measured Concentrations of the Test Item Disperse Blue ANT

Sampling date

2015-03-17

Start of the exposure

0 hours

Start of analysis

2015-03-17

Nominal

loading rate

of the test item

Disperse Blue ANT

[%]

[mg/L]

Meas. conc.

[mg/L]

100*

5.00*

< LOQ

 50.0

2.50

Considered to be also < LOQ,
due to the results of the
saturated solution

 25.0

1.25

 12.5

0.625

   6.25

0.313

Control

< LOQ

Meas. conc.= measured concentration of the test item, dilution factor taken into account

*                    = saturated solution

LOQ             = limit of quantification of theanalytical method(0.5 mg/L of the test item)

Validity criteria fulfilled:
yes
Conclusions:
At the saturated solution with a nominal loading of 5.00 mg/L of the test item Disperse Blue ANT and at any of the tested dilution levels, no significant effects were observed on Daphnia magna.

According to OECD Guidance Document No 23 (2000) if an effect cannot be detected in a test with a saturated solution the result has to be reported as no
toxic effects at saturation. Therefore, exposures are 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 saturated solution.
Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item Disperse Blue ANT were determined according to EPA OPPTS Draft Guideline 850.1010 (1996) and OECD 202 (2004) which is equivalent to the Council Regulation (EC) No.440/2008 Method C.2 (2008). The study was performed from 2015-03-16 to 2015-03-19, with the definitive exposure phase from 2015-03-17 to 2015-03-19, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, D-31157 Sarstedt, Germany.

 

The study was conducted under static conditions over a period of 48 hours with a saturated solution with a nominal loading of 5.00 mg/L of the test item Disperse Blue ANT and further dilution levels in a geometric series with a separation factor of 2 (nominal loading rates of 0.313 to 2.50 mg/L).

 

The test item Disperse Blue ANT is a dark green powder and has a water solubility of 0.13 mg/L. The saturated solution with a nominal loading of 5.00 mg/L of the test item was prepared with dilution water one day before the start of the exposure (at -24 hours) and was used for testing and as stock solution for the preparation of all further tested dilution levels. A test item dispersion of 5 mg/L was stirred with approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, the test item dispersion was filtered with a membrane filter (0.2 µm, RC,Macherey-Nagel) to achieve the saturated solution. Further dilution levels were prepared by diluting the saturated solution with dilution water.The tested solutions were visually clear throughout the exposure period.Twenty daphnids were exposed to each concentration level and the control.

 

The concentrations of the test item Disperse Blue ANT in the saturated solution and in the control were analytically verified via HPLC-UV at the start of the exposure (0 hours).

 

The measured concentrations of the test item at the start of the exposure (0 hours) were below the LOQ of 0.5 mg/L of the test item in the saturated solution and in the control. Concentrations of the test item in all tested loading levels were considered to be all below the LOQ throughout the exposure. Due to this fact, analytical monitoring of the other dilution levels at the start of the exposure and the analytical monitoring of all loading levels at the end of the exposure was not carried out in agreement with the sponsor.

 

According to OECD Guidance Document No 23 (2000) if an effect cannot be detected in a test with a saturated solution the result has to be reported as no toxic effects at saturation. Therefore,exposures are 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 saturated solution.

 

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

 

 

 

At the saturated solution with a nominal loading of 5.00 mg/L of the test item

Disperse Blue ANT and at any of the tested dilution levels,

no significant effects were observed on Daphnia magna.

 

 

 

EL-Values, NOEL and LOEL

(based on the nominal loading rate of the test item in the saturated solution)

Endpoints

 

 

Test duration

[h]

Disperse Blue ANT

Nominal loading rate

of the test item in the saturated solution

[mg/L]

EL10/ EL50/ EL100

24

> 5.00

48

> 5.00

NOEL

48

  5.00

LOEL

48

> 5.00


 

Description of key information

No adverse effects noted at the limit of solubility and far beyond (OECD 202, structural analogue substance).

Key value for chemical safety assessment

Additional information