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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 2017-04-09 to 2017-04-13, with the definitive exposure phase from 2017 04 10 to 2017-04-12
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)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Determination of the test item
The saturated solution and the control were analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of the saturated solution and analyzed.
At the end of the exposure (48 hours), samples of the old media were taken for analysis from additional replicates, which were prepared with test media, but without daphnids. These additional replicates were incubated under test conditions until sampling.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or
initially measured concentration.
Vehicle:
no
Details on test solutions:
Limit concentration
A saturated solution with a nominal loading of 20.0 mg/L of the test item was tested in a limit test. The maximum water solubility was determined to be 20 (± 3) mg/L in an OECD 105 study.

Preparation of the saturated solution
The saturated solution (20.0 mg/L test item were weighed out) was prepared with dilution water (see Table 2) one day before the start of the exposure (at -24 hours). The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used as a limit concentration in the test. During filtration, the filter was always kept covered.

Control
Dilution water without test item incubated under the same conditions as the test group
Test organisms (species):
Daphnia magna
Details on test organisms:
Test system
Daphnia magna STRAUS (Clone 5)

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
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

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.

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.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
see any other information on material and methods incl. tables


Test temperature:
see any other information on material and methods incl. tables
pH:
see any other information on material and methods incl. tables
Dissolved oxygen:
see any other information on material and methods incl. tables





Conductivity:
see any other information on material and methods incl. tables
Nominal and measured concentrations:
Nominal: 100% of the saturated solution
Geometric mean measured test item concentrations: 6.73 mg/L.
Details on test conditions:
Test method
The study was performed under static conditions with a renewal of the test solutions after 24 hours.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL in each test replicate (see ‘Application’)

Dilution water
Same as culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each loading level and the control.

Age of the daphnids
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure 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.

Acclimatization
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate was 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 a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.



Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
other: EC10/50/100
Effect conc.:
> 6.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
other: EC10/50/100
Effect conc.:
> 6.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 04-06 to 2017-04-07.

EC50-Value (with 95% confidence limits) of the Reference Item Potassium dichromate based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.72 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000)
0.6 - 2.1 mg/L, acc. to OECD 202 (2004)
95% confidence limits 1.34 - 1.93 mg/L


Reported statistics and error estimates:
Methods of evaluation
The effects of the saturated solution were deduced directly from the observed immobilization rates. The effect levels (EC10 / 50 / 100) given are based on the geometric mean measured concentration.

EC-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.

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 Ratesafter 24 and 48 hours ofExposure in the Definitive Test

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

Acid Blue 324

Geometric mean

measured test item concentration

[mg/L]

IMMOBILIZATION [%]

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

6.73*

0

0

0

0

0

0

0

0

0

0

Control

0

0

0

0

0

0

0

0

0

0

* = saturated solution

 

 Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

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

Acid Blue 324

Geometric mean

measured test item concentration

[mg/L]

Number of immobile Daphnids / Total number of Daphnids

24 hours

48 hours

Replicates

Replicates

1

2

3

4

MV

1

2

3

4

MV

6.73*

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

* = saturated solution

 

 

Additional Observations during the Definitive Test

 

The saturated solution was dark blue and visually clear during the exposure period.

Validity criteria fulfilled:
yes
Conclusions:
In the saturated solution prepared with a nominal loading of 20.0 mg/L of the test item Acid Blue 324, which corresponds to a geometric mean measured test item concentration of 6.73 mg/L, no effects on Daphnia magna were observed.
Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of a saturated solution with a nominal loading of 20.0 mg/L of the test item AcidBlue 324 were determined at the test facility according to OECD 202 (2004) from 2017-04-09 to 2017-04-13, with the definitive exposure phase from 2017‑04‑10 to 2017-04-12.

The limit test was conducted under static conditions over a period of 48 hours.The saturated solution was dark blue and visually clear during the exposure period. Twenty daphnids, divided into 4 replicates with 5 daphnids each, were exposed to the saturated solution and the control.

The concentration of the test item AcidBlue 324 was analytically verified via HPLC-DAD in fresh media at the start of the exposure (0 hours) and in old media at the end of the exposure (48 hours) in the saturated solution and in the control.

The measured concentration in the saturated solution was 6.81 mg/L at the start of the exposure (0 hours) and 6.64 mg/L at the end of the exposure (48 hours), representing 97% of the initially measured test item concentration.The geometric mean measured test item concentration is 6.73 mg/L.

The measured test item concentrations were within ± 20% of the initially measured concentration. This indicates that the test item concentration was successfully maintained for the duration of the test. The validity criteria of the test guideline were fulfilled.

 

 EC10 / 50 / 100-Values

(based on the geometric mean measured concentration of the test item)

Effect concentrations

Test

duration

[hours]

Acid Blue324

Geometric mean measured concentration of the test item

[mg/L]

EC10 / 50 / 100

24

> 6.73

48

> 6.73

 

 

Description of key information

In the saturated solution prepared with a nominal loading of 20.0 mg/L of the test item Acid Blue 324, which corresponds to a geometric mean measured test item concentration of 6.73 mg/L, no effects on Daphnia magna were observed. No effect occurred up to the limit of solubility in the test medium. The EC50 is > solubility limit and therefore not considered acutely toxic to aquatic invertebrates.

Key value for chemical safety assessment

Additional information

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