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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:
Start : 21 May 2012 Completion : 20 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with no significant deficiencies
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the
schedule below. In addition, glass wool containing undissolved residue was kept for possible analysis.
Frequency at t=0 h and t=48 h
Volume 2 ml from the approximate centre of the test vessels
Storage Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 2 ml were taken for possible analysis. If not used, these samples
were stored in a freezer for a maximum of three months after delivery of the draft report, pending on
the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
Preparation of
test solutions started with individual loading rates ranged between 0.1 and 100 mg/l.
A 2-day period of magnetic stirring was applied to obtain maximum dissolution of the substance in
medium. After a 1-hour settlement period the Water Accommodated Fractions (WAFs) were siphoned
off through glass wool and used as test concentrations. All test solutions, besides the highest WAF in
the combined limit/range-finding test which was slightly hazy, were clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally
accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood,
showing no signs of stress such as mortality >20%,
presence of males, ephippia or discoloured animals and
there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnids with an age of < 24
hours, from parental daphnids of more than two weeks old.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Macro salts:
CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Test temperature:
The temperature of the test medium was 20.5°C at the start of the test. The temperature continuously
measured in a temperature control vessel varied between 19.9 and 20.8°C during the test, and
complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
Day 0: 7.8 - 8
Day 2: 7.9-8
Dissolved oxygen:
Day 0: 8.5 - 9.1
Day 2: 7.9 - 9.0
Salinity:
NA
Nominal and measured concentrations:
See Table
Details on test conditions:
Test duration -- 48 hours
Test type -- Static
Test vessels -- 100 ml, all-glass
Medium -- Adjusted ISO medium
Number of daphnids -- 20 per concentration
Loading -- 5 per vessel containing 80 ml of test solution
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
28 h
Dose descriptor:
EC50
Effect conc.:
> 0.27 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
A table of mortality is presented in the section below.
In the WAF prepared at 4.6 mg/l all daphnids were immobilized at the end of the test. Microscopic
observation of the immobile daphnids showed test substance being attached to the bodies of the
immobilised daphnids. Therefore, it was assumed that this effect was mechanical rather than caused
by toxicity of the test substance as the concentration measured in the highest WAF was higher but
caused less effect.
At the highest concentration 20% of daphnids were immobilised. Also in this concentration test
substance was observed on daphnia bodies. Therefore, the observed effect could be also mechanical
rather than of toxic origin.
Results with reference substance (positive control):
See Table.
Reported statistics and error estimates:
EC50 could not be calculated due to insufficient mortality

Acute immobilisation of daphnids after 24 and 48 hours in the final test

Test substance1

WAF prep. at

(mg/l)

Vessel

number

 

Number

Daphnia

exposed

Response at 24 h

Response at 48 h

 

number

Total

%

 

number

Total

%

control

 A

 B

C

D

5

5

5

5

0 [2]

0

0

0

0

0

0

0

0

0

4.6 (0.033)

 A

 B

C

D

5

5

5

5

0

5

1

2

40

5*

5

5

5

100

10 (0.27)

 A

 B

C

D

5

5

5

5

0

0

1

0

5

0

0

0

0

0

22 (0.035)

 A

 B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

46 (0.0071)

 A

 B

C

D

5

5

5

5

0

0

0

0

0

0

0

0

1

5

100 (0.16)

A

B

C

D

5

5

5

5

0

0

0

1

5

0

2*

0

2

20

1.di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)

() – average exposure concentration

[] - number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

* - test substance observed stuck to daphnia (microscopic observation).

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium

dichromate:

 

Concentration

(mg/l)

Number

Exposed

% immobile

 

Expected response (%)

After 48 hours1

 

 

24 h

48 h

minimal

maximal

control

20

0

0

0     

102

 

 

 

 

 

 

0.10

20

0

0

0  

10

0.18

20

0

0 

0    

10

0.32

20

0

85

0       

30

0.56

20

75

95 

0     

100

1.0

20

100

100

40     

100

1.8

20

100

100 

100     

100

1Based on historical data of the previous years (n>60).

2A maximum response of 10% does not invalidate the results of the test.

Validity criteria fulfilled:
yes
Conclusions:
Due to the very low solubility of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) in test medium, concentrations that might be toxic to Daphnia magna could not be reached. The 48-hour EC50 > 0.27 mg/L (measured).
Executive summary:

An Acute Toxicity Study in Daphnia magna was conducted with di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA). The study procedures described in this report were based on the OECD guideline No. 202, 2004. The batch of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) tested was brown highly viscous liquid and a UVCB (treated as 100% pure) substance. The test substance was not completely soluble in test medium at the loading rates initially prepared. A final test was performed based on the results of a combined limit/range-finding test. Preparation of test solutions started with individual loading rates ranged between 0.1 and 100 mg/l. A 2-day period of magnetic stirring was applied to obtain maximum dissolution of the substance in medium. After a 1-hour settlement period the Water Accommodated Fractions (WAFs) were siphoned off through glass wool and used as test concentrations. All test solutions, besides the highest WAF in the combined limit/range-finding test which was slightly hazy, were clear and colourless. Twenty daphnids per group (5 per vessel, 4 vessels) were exposed to a control and WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/l in the final test. Total exposure time was 48 hours and samples for analytical confirmation of the exposure concentrations were taken at the start and at the end of the test. Analysis of the samples taken at the start of the final test showed measured concentrations being highly variable and not directly related to the applied loading rates. This was probably caused by the very low solubility of the substance in the medium and the fact that all concentrations were prepared separately. The concentration measured in the WAF prepared at 100 mg/l was 0.180 mg/l, which is close to the water solubility of the test substance (0.23 mg/l, WIL Research Europe project 498817). At the end of the test the actual concentrations were between 52 and 77% of initial. Therefore, the effect parameters were expressed in terms of average exposure concentration. The highest average exposure concentration was measured in the WAF prepared at 10 mg/l and corresponded to 0.27 mg/l. The study met the acceptability criteria prescribed by the protocol and was considered valid. For Daphnia magna exposed to di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) the 48h-EC50 was beyond the range tested, i.e. exceeded the average exposure concentration of 0.27 mg/l. Due to the very low solubility of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) in test medium, concentrations that might be toxic to Daphnia magna could not be reached. The effects observed at the higher measured concentrations of the test substance were mechanical rather than toxic.

Description of key information

The 48-hour EC50 > 0.27 mg/L (measured).
Due to the very low solubility of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) in test medium,

concentrations that might be toxic to Daphnia magna could not be reached.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.27 mg/L

Additional information