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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:
2016-04-19 to 2016-04-21
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 limit concentration (pH-neutralised and un-neutralised) and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (48 hours). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented.

Sampling for the analyticalmonitoring
At the start of the exposure (0 hours), sampling was carried out after preparation of the limit concentration (pH-neutralised and un-neutralised).
At the end of the exposure (48 hours), samples were taken directly from the test vessels.

Quality criteria for the analytical monitoring
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 limit concentration of the test item of 133 mg/L was tested pH neutralised and un-neutralised.

Preparation of the limit concentration
The limit concentration (133 mg/L of the test item were weighed out) was freshly prepared with dilution water (ISO test water as specified below) before the start of the exposure (0 hours).

Treatment T
he limit concentration was mixed thoroughly by manual agitation.

pH-neutralised concentration
In addition to the un-neutralised limit concentration of 133 mg/L, an appropriate aliquot of the limit concentration was adjusted to pH 7 ± 0.2 by the addition
of 1 M HCl prior to the start of the exposure and tested under the same conditions as the un-neutralised concentration.

The limit concentration (pH-neutralised and un-neutralised) is based on the results of a non-GLP preliminary range finding test conducted at the test facility
under static conditions. For results, see section 6.1.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test method
The study was performed with a static test design, since the test item concentrations in the non-GLP range finding test indicated sufficient stability over a
period of 48 hours. The limit concentration was tested pH-neutralised and un-neutralised to distinguish toxic effects of the test item from pH effects.


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. 20 µEm-2  s-1 (max. 1340 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. The composition of the culture medium
is presented

Culture feeding
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.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany



Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)
Component Concentration [mg/L]
CaCl2 x 2 H2O 176*
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
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8
* = original recipe: 293.8 mg/L, modified to achieve a total water hardness of
160 to 180 mg CaCO3/L


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
Dilution water dated:

0 hours: 2016-04-19 Total hardness [mg CaCO3/L]: 258

Test temperature:
18 - 22°C, constant within ± 1°C
2016-04-19 : 20.8 °C
pH:
Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the limit concentration (pH-neutralised and un-neutralised) and the control)

Nominal test item
concentrations
[mg/L] 0 hours
pH-value Dissolved O2 concentration [mg/L]

1331)
(pH-neutralised) 7.00 8.62
133 9.27 8.72
Control 8.00 8.78
1) = adjusted from pH 9.27 to pH 7.00 by addition of 1 M HCl


Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of the limit concentration (pH-neutralised and un-neutralised) and the control))

Nominal test item
concentrations

[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
1331)
(pH-neutralised) 7.73 7.72 7.70 7.70 7.94 8.16 8.38 8.41
133 7.91 7.93 7.97 7.96 8.49 8.38 8.48 8.29
Control 7.73 7.74 7.80 7.81 8.42 7.91 8.24 8.33
1) = adjusted from pH 9.27 to pH 7.00 by addition of 1 M HCl


Dissolved oxygen:
Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the limit concentration (pH-neutralised and un-neutralised) and the control)

Nominal test item
concentrations
[mg/L] 0 hours
pH-value Dissolved O2 concentration [mg/L]

1331)
(pH-neutralised) 7.00 8.62
133 9.27 8.72
Control 8.00 8.78
1) = adjusted from pH 9.27 to pH 7.00 by addition of 1 M HCl


Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of the limit concentration (pH-neutralised and un-neutralised) and the control))

Nominal test item
concentrations

[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
1331)
(pH-neutralised) 7.73 7.72 7.70 7.70 7.94 8.16 8.38 8.41
133 7.91 7.93 7.97 7.96 8.49 8.38 8.48 8.29
Control 7.73 7.74 7.80 7.81 8.42 7.91 8.24 8.33
1) = adjusted from pH 9.27 to pH 7.00 by addition of 1 M HCl
Details on test conditions:
Test method
The study was performed with a static test design, since the test item concentrations in the non-GLP range finding test indicated sufficient stability over a
period of 48 hours. The limit concentration was tested pH-neutralised and un-neutralised to distinguish toxic effects of the test item from pH effects.

Test duration
48 hours

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

Test volume
20 mL

Dilution water ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123


Number of daphnids
20 daphnids, divided into 4 replicates, each with 5 daphnids,
and replicates were used for the limit concentration (pH-neutralised and un neutralised) and the control.

Age of the daphnids at the start of the exposure
2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 22 hours
before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an acclimatisation phase of at least 2 hours in the dilution water. No first brood progeny was used for the test.

Acclimatisation
At least 2 hours in dilution water

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 with ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 20 µEm-2  s-1 (max. 1340 lx)

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

Feeding
The daphnids were not fed during the study.





Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
> 133 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
> 133 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 133 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 133 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 133 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 133 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Results with reference substance (positive control):
The percentage of 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: 1.95 mg/L (Cl: 1.14 - 3.30 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the required 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:
Methods of evaluation
The effects of the limit concentration (un-neutralised) were deduced directly from the observed immobilisation rates. Since the measured concentrations were within ± 20% of the nominal value, the effect levels (EC10 / 50 / 100) were based on the nominal concentration of the test item Genamin DMG 75.

EC-values and statistical analyses for reference item
An EC50-value was calculated only 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

 

Water Quality Parameters of the Dilution Water at the Start of the Exposure (0 hours)

Dilution water

dated:

 

pH-value

 

 

Dissolved

O2concentration

[mg/L]

Temperature

 

[°C]

Conductivity

 

[µS/cm]

Total hardness

 

[mg CaCO3/L]

2016-04-19

8.00

8.78

20.8

647

258

 

Validity criteria fulfilled:
yes
Conclusions:
In the limit concentration 133 mg/L of the test item Genamin DMG 75,
which is equivalent to 100 mg/L pure compound (100% (w/w) minus water content of 24.8% (w/w)), no effects on Daphnia magna were observed.


Executive summary:

    

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the limit concentration 133 mg/L of the test item Genamin DMG 75 , which is equivalent to 100 mg/L pure compound (100% (w/w) minus water content of 24.8% (w/w)), were determined according to OECD 202 (2004) from 2016-04-19 to 2016-04-21 at the test facility.

The study was conducted under static conditions over a period of 48 hours. Additionally, the limit concentration was tested pH-adjusted.The pH adjustment reflects the actually existing environmental conditions in the buffered aquatic systems.

The test item Genamin DMG 75is a slightly yellowish solution, which is soluble in water.

The limit concentration of 133 mg/L of the test item was mixed thoroughly by manual agitation. Additionally, the pH of the limit concentration wasadjusted from pH 9.27 to a pH of 7.00 by addition of 1 M HCl and tested under the same conditions as the not adjusted limit concentration.Thelimit concentration (pH-adjusted and un-adjusted)was visually clear throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the limit concentration (pH-adjusted and un-adjusted) and the control.

The concentration of the test item Genamin DMG 75were analytically verified via LC-MS/MS at the start of the exposure (0 hours) and at the end of the exposure (48 hours) in the limit concentration (pH-adjusted and un-adjusted) and in the control. Details of the analytical method are presented in section11.

The measured concentrations of the test item were in the range of 91 to 95% of the nominal values at the start (0 hours) and at the end of the exposure (48 hours).The analytical results arepresented inTable 6.

The measured test item concentrations were all within ±20% of the nominal concentration. This indicates that the test item concentration was successfully maintained for the duration of the test. The effect levels based on the nominal concentration of test item Genamin DMG 75 are given .

The validity criteria of the test guideline were fulfilled.

 

 

 

In the limit concentration133 mg/Lof the test itemGenaminDMG 75,
which is equivalent to 100 mg/L pure compound (100% (w/w) minus water content of 24.8% (w/w)),no effects onDaphnia magnawere observed.

 

 

 

  EC10-, EC50-and EC100-Values

(based on the nominal concentration of the test itemGenaminDMG 75)

Effect levels

 

 

Test

duration

[hours]

Toxicity endpoint based on the

nominal concentration the test item [mg/L]

pure compound*

[mg/L]

EC10 / 50 / 100

24

> 133

> 100

48

> 133

> 100

*    = (100% (w/w) minus water content of 24.8% (w/w)

Description of key information

In the limit concentration 133 mg/L of the test item Genamin DMG 75,

which is equivalent to 100 mg/L pure compound (100% (w/w) minus water content of 24.8% (w/w)), no effects on Daphnia magna were observed.

Key value for chemical safety assessment

Additional information