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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:
February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 202 guideline study carried out under GLP
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
10 mL of all test concentrations were sampled for analysis at the start and end of the test and were subsequently transferred to HPLC vials and diluted as required with DSW.
Vehicle:
no
Details on test solutions:
Preparation of solutions
The test chemical was known to dissolve readily in the test media. A stock of approximately 100 mg/L was made by accurate weighing of the test chemical addition and subsequent moderate to fast stirring until a homogeneous solution was achieved. pH adjustment was not required. The test chemical was then pipetted (whilst stirring the stock solution) in the correct volumes to volumetric vessels that were subsequently made up to volume with test medium and stirred thoroughly generating the test concentrations indicated below.
Test organisms (species):
Daphnia magna
Details on test organisms:

Source and maintenance of the test organisms
The test animals were taken from a Daphnia magna stock, cultured in conformity with Stan¬dard Operation Procedure (E 1). The animals used in the test were less than 24 hours old were obtained from parent animals aged between 2-4 weeks. Daphnia were originally obtained from NOTOX B.V. Hambakenwetering 7 5231 DD ‘s-Hertogenbosch.The Netherlands.

Quality control of the test organisms
The sensitivity of the Daphnia was checked by performing an inhibition test with a reference compound (potassium dichromate) twice a year. The sensitivity was tested for compliance with the guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
per liter of de-ionized water: 200 mg of CaCl2•2H2O, 180 mg of MgSO4•7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3
Test temperature:
19.95 to 21.25 °C
pH:
8.1 to 8.9
Dissolved oxygen:
8.9 to 9.0 mg/L
Salinity:
no data
Nominal and measured concentrations:
Nominal (mg/L) 0.6; 1.4; 3.0; 6.8; 15
Measured (mg/L) 0.3; 0.4; 1.6; 4.2; 9.7
Details on test conditions:
Dilution Water
The test medium was Dutch Standard Water (DSW), having a pH of approximately 8.2, and a conductivity between 550 and 650 s/cm, con¬taining per liter of de-ionized water: 200 mg of CaCl2•2H2O, 180 mg of MgSO4•7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 (Standard Operation Procedure O1) . The guideline criteria requires the CaCO3 content to be between 10 and 250 mg/L (approximately 1-14 ºdH). Hardness in the test water was measured once at the start of the study using the appropriate Dr Lange test kit. This was validated by analyzing a CaCl2 Standard. The dilution was saturated with oxygen before the start of the test. This is done by thorough aeration for at least 30 minutes before use.

General test principles and procedures
The test was performed as a static test for 48 hours. 20 animals divided into 4 batches of 5 animals was tested at each of 5 test concentrations and in the control. Those animals which are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be im¬mobile. The number of animals being trapped at the surface was determined. These animals were not regarded as im¬mobile and were made to re-submerge. In summary the daphnids were randomly placed in the test fluids and the test vessels were placed in a random manner within each group. The test vessels were not aerated during the test and the animals were not fed. The test was inspected at 0, 24 and 48 hours.

Two definitive studies were carried out using identical methods.

Test concentrations
Following a preliminary test, the definitive test was conducted at 0.6, 1.4, 3.0, 6.8 and 15 mg/L.

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
6.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
4.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
9.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
no abnormal observations
Results with reference substance (positive control):
The EC50 value of the reference compound, potassium dichro¬mate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).
Reported statistics and error estimates:
The Dunnetts test was used for the NOEC/LOEC determination and the Trimmed spearman karber method was used for the EC50 determination. Normality and equality of variance of the data could not be confirmed. Considering the single mortality occurring at the NOEC concentration the NOEC is expected to be reliable and indeed representative of no significant effect despite the results of the auxiliary tests. All calculations were conducted using the ToxCalc 5.023 software

Numbers of mobile Daphnia

Conc mg/l

 0 hours

 24 hours

 48 hours

Control I

 5

 5

 5

II

 5

 5

 5

III

 5

 5

 4

IV

 5

 5

 4

Total

 20

 20

 18

0.6         I

 5

 5

 5

II

 5

 5

 5

III

 5

 5

 5

IV

 5

 5

 5

Total

 20

 20

 20

1.4         I

 5

 5

 5

II

 5

 5

 5

III

 5

 5

 4

IV

 5

 5

 4

Total

 20

 20

 18

3.0         I

 5

 5

 5

II

 5

 5

 5

III

 5

 5

 5

IV

 5

 5

 5

Total

 20

 20

 20

6.8         I

 5

 5

 5

II

 5

 5

 4

III

 5

 5

 5

IV

 5

 5

 5

Total

 20

 20

 19

15          I

 5

 5

 0

II

 5

 5

 0

III

 5

 5

 0

IV

 5

 5

 2

Total

20

20

2

Validity criteria fulfilled:
yes
Remarks:
(1) Immobilization in the control did not exceed 10% (2) Oxygen concentration did not fall below 3 mg/L at any point during the study, (3) No daphnids were trapped on the surface for any length of time in the second definitive test
Conclusions:
The EC50 (48h) was calculated as 6.7 mg/l. (Measured Geometric Mean)
Executive summary:

Summary

In order to predict the effects of 1,1,3,3-Tetramethylbutyl Hydroperoxide in an aquatic environment, an acute toxicity test to Daphnia magna was conducted in accordance with OECD test guidelines and with the OECD Principles of Good Laboratory Practice.

The toxicity of the test chemical and any formed degradation products to juvenile daphnia was determined in a static system over an exposure period of 48 hours. The following concentrations were tested 0.6, 1.4, 3.0, 6.8 and 15 mg/L. The NOEC was determined as 4.21 mg/L and the LOEC was determined as 9.72 mg/L (Measured Mean Concentration). The EC50 (48h) was calculated as 6.7 mg/l. (Measured Mean).

Chemical analysis was conducted at the start and at the end of the study. Analytical quality criteria were met. Geometric measured mean concentrations were calculated for use in the end point calculations.

The following quality criteria were met in the final definitive study:

  • Immobilization in the control did not exceed 10%
  • Oxygen concentration did not fall below 3 mg/L at any point during the study
  • The EC50 value of the reference compound, potassium dichromate, was in the range of 0.25-2.0 mg/L (Documented as part of GLP laboratory maintenance).
  • No daphnids were trapped on the surface for any length of time.
  • The analytical validity criteria set in the study plan were met.

The following quality criterion was not met:

The test substance did not remain stable during the test.

Investigation of the effects of the non readily biodegradable degradation products would benefit further risk assessment of this substance.

Description of key information

One OECD 202 GLP study is available with a 48 h EC50 of 6.7 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
6.7 mg/L

Additional information

One OECD 202 GLP study " 5809-08-5, Short-term toxicity to aquatic invertebrates, Kean, 2012, RS" with analytical monitoring is available which meets all the quality criteria. As the test substance did not remain stable during the test, the EC50 was calculated based on geometric mean values.