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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 October 2013-30 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study has been performed according to OECD and/or EC guidelines and according to GLP principles
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
(2014)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Singular samples for analysis were taken from all test concentrations and the controls.
Sampling:
Frequency:At the start, after 7, 15 and 21 days.
Volume: 2.0 ml
Storage: Not applicable, all sample were analysed on the day of sampling.
Vehicle:
yes
Details on test solutions:
Stock solutions were prepared in Acetone (Merck, Darmstadt, Germany, 99.8%) at weekly intervals at concentrations exceeding the target concentrations by a factor of 10,000. No other treatment than careful shaking was necessary to completely dissolve the stocks in Acetone resulting in clear and colourless solutions. Note that weighing and formulation was as much as possible protected from light and stocks were wrapped in aluminum foil during dosing.
Exact volumes of the 2,6-di-tert-butylphenol stock solutions were dosed using diluters with a 1.0 ml syringe into the various mixing vessels. M7 medium was prepared by first dosing ISO-medium with a flow-meter set at 5 l/h in a collection vessel. From this collection vessel the ISO-medium was transferred to each of the 7 mixing vessels using a peristaltic pump set at 500 ml/h. An algae suspension containing M7-salts was also transferred to each of the 7 mixing vessels using a peristaltic pump set at 208 ml/h. The dosed volumes of test substance stocks, M7-medium and algae were mixed under continuous stirring in a mixing vessel. From this vessel it entered the test vessels containing the daphnids. The whole system was checked daily. Note that the mixing vessels were protected from light by aluminum foil.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
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: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
Start of each batch: With neonate daphnids, i.e. less than 3 days old by placing them individually in 50 ml M7-medium.
Maximum age of the cultures: 4 weeks
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae
Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
Medium:M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
between 161 and 196 mg calcium carbonate per litre,
Test temperature:
19-21°C
pH:
7.2 - 7.8
Dissolved oxygen:
4.6 - 9.0 mg/l
Nominal and measured concentrations:
Nominal concentrations: Target concentrations of 0.046, 0.10, 0.22, 0.46 and 1.0 mg/l.
Measured concentrations: average measured concentrations of 0.035, 0.086, 0.19, 0.34 and 0.60 mg/l.
Details on test conditions:
Test duration: 21 days
Test type: Flow-through, with continuous renewal of test media
Test vessels:Stainless steel, covered with a Perspex plate
Medium: M7
Experimental design: For each test group the system comprised of one stainless steel vessel of ~1.5 litre with 4 mesh containers of stainless steel. At the start of the test five neonate daphnia were placed in each mesh.

OTHER TEST CONDITIONS
Light: 16 h photoperiod daily;
intensity at the start: 194-220 lux
intensity at the end: 226-259 lux
Feeding: A Chlorella pyrenoidosa suspension was continuously added as feed for the daphnids. The daily ration theoretically corresponded to 0.4 mg C/Daphnia/day after correction for the waste of algae that was inherent to the use of a flow-through design. This daily ration provided is twice the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.
Introduction of daphnids : After stabilisation of the flow-through system.

EFFECT PARAMETERS MEASURED
21-day LC50 (parental mortality)
21-day NOEC for parental mortality
21-day EC50 for reproduction
21-day LOEC for reproduction
21-day NOEC for reproduction
21-day LOEC for parental body length
21-day NOEC for parental body length

TEST CONCENTRATIONS
Rangefinding (semi static): The concentrations of 2,6-di-tert-butylphenol tested in the range-finding test corresponded to 1.0, 10 and 100% of a Water Soluble Fraction (WSF) prepared at a loading rate of 1.0 mg/l.
Final (flow trough):Target concentrations of 0.046, 0.10, 0.22, 0.46 and 1.0 mg/l a control and solvent-control
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parental
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
parental
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.086 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.086 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Parental body lenght
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.035 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
parental body lenght
Details on results:
MORTALITY OF PARANTAL DAPHNIDS:
Blank-control: 15%
Solvent-control: 0%
0.035 mg/l: 5%
0.086 mg/l: 25%
0.19 mg/l: 35%
0.34 mg/l: 40%
0.60 mg/l: 100%

MEAN NUMBER OF OFFSPRING:
Blank-control: 97.6 ± 11.2
Solvent-control: 126.1 ± 23.6
0.035 mg/l: 176.5 ± 20.0
0.086 mg/l: 87.7 ±46.6
0.19 mg/l: 83.8 ± 62.1
0.34 mg/l: 0.6 ± 0.8

AVERAGE BODY LENGHT OF PARENTAL DAPHNIDS :
Blank-control: 4.03 ±0.15 mm
Solvent-control: 3.96 ± 0.20 mm
0.035 mg/l: 4.11 ± 0.16 mm
0.086 mg/l: 3.74 ± 0.29 mm
0.19 mg/l: 3.91 ± 0.15 mm
0.34 mg/l: 3.61 ± 0.23 mm

Type and number of morphological abnormalities: not observed
Type and number of behavioural abnormalities: not observed
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Statistical analysis:

Total reproduction and body length on day 21 were tested for normality and for homogeneity of variance and statistically tested using the Williams Multiple Sequential t-test (reproduction and growth). Parental survival on day 21 was statistically tested using the Fisher`s Exact Binomial Test with Bonferroni Correction.
All testing was performed using ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

LOEC and NOEC
The overall threshold level of effect and the overall NOEC were determined on basis of the statistical analyses.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study 2,6-di-tert-butylphenol did not affect reproduction, survival or growth of Daphnia magna at an average measured concentration of 0.035 mg/l after 21 days of exposure (NOEC).
Executive summary:

Daphnia magna, 21-day reproduction study with 2 ,6-di-tert-butylphenol.

The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition, the procedures were designed to meet the test methods and validity criteria of the ISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

The batch of 2,6-di-tert-butylphenol tested was a colourless crystal solid with a purity of 99.7% and the substance was completely soluble in test medium at the target concentrations tested in the main study.

The reproduction test was performed using a flow-through system with target concentrations of 0.046, 0.10, 0.22, 0.46 and 1.0 mg/I, which was based on the result of a preceding semi-static range-finding test. A blank- and solvent-control (Acetone; 100 pIll) were also included.

In a flow-through system the dilution water (M7 medium) was dosed separately from the test substance stock solutions into mixing vessels, 2,6-di-tert-butylphenol stock solutions in Acetone were prepared at weekly intervals. The dosed volumes and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily. The experimental set-up included stainless steel vessels with 4 stainless steel mesh containers per test group. Each container contained five neonate (<24 h old) Daphnia magna. The study duration was 21 days and the test solutions were renewed at a flow rate of 0.7 litres/hour. The daphnids were fed continuously with Chlorella pyrenoidosa (-0.4 mg organic C per daphnid per day). The condition of the parental daphnids was recorded daily and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

During the study samples for analyses were taken at the start of exposure and on days 7, 15 and 21. Analyses showed that measured concentrations were fairly stable during the test period and that they generally ranged between~ 70 and 90% relative to the target concentrations. The average measured concentrations were 0.035, 0.086, 0.19, 0.34 and 0.60 mg/I.

Three of the twenty introduced parental daphnids died during the test period in the blank-control, while no daphnids died in the solvent-control. Only 1 parent died in the lowest concentration of 0.035 mg/I. The numbers of dead parents increased with increasing target concentrations. The total number of daphnia found dead per group were 5, 7, 8 and 20 at respectively 0.086, 0.19, 0.34 and 0.60 mg/I. Note that 3 daphnia could not be recovered in the highest test group during the first three days. These daphnia were regarded as having died for conservative reasons and because all other daphnia in this group died within 6 days.

The average cumulative numbers of young per female in the blank-control and solvent-control were 98 and 126, respectively. The average cumulative reproduction numbers at the study end were 177, 88, 84, 1 and 0 for respectively 0.035, 0.086, 0.19, 0.34 and 0.60 mg/I. No significant numbers of aborted eggs or immobile young were observed at any of the concentrations tested.

Parental daphnia were slightly bigger in the lowest test group when compared to the treatment control, while the size of the parents exposed to 0.086, 0.19 and 0.34 mg/I were reduced by 5.6, 1.3 and 8.8%, respectively. At the highest test concentration body length could not be determined since all parents had died.

Following deviations to the protocol occured during the study: relatively low oxygen concentrations (down to 4.6 mg/L) in some of the test groups at the study start,(thereafter the minimum DO concentration was 6.6 mg/L throughout the study), accidental loss of three parents in the highest test group and finally an observed difference in effects at the highest test group between the semi-static range-finder and the final flow-through study. These were considered to have no effect on the final outcome or the integrity of the study.

It was concluded that all test conditions were maintained within the limits prescribed by the protocol and the test was considered valid.

Effect parameters obtained in this study are summarized in the table below.

Parameter I

Concentration
2,6-di-tert-butylphenol
Average (mg/I)

21-day LC5o (parental mortality)

0.23

21-day NOEC for parental mortality

0.035

21-day EC50 for reproduction

0.14

21-day LOEC for reproduction

0.086

21-day NOEC for reproduction

0.035

21-day LOEC for parental body length

0.086

21-day NOEC for parental body length

0.035

1All parameters determined using Toxrat

Under the conditions of the present study 2,6-di-tert-butylphenol did not affect reproduction, survival or growth of Daphnia magna at an average measured concentration of 0.035 mg/I after 21 days of exposure (NOEC).

Description of key information

2,6-di-tert-butylphenol did not affect reproduction, survival or growth of Daphnia magna at an average measured concentration of 0.035 mg/L after 21 days of exposure (NOEC).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.035 mg/L

Additional information

A Daphnia magna, 21-day reproduction study with 2,6-di-tert-butylphenol is available. The study procedures described in this report were based on the OECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition, the procedures were designed to meet the test methods and validity criteria of the ISO International Standard 10706, 2000, the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

It was concluded that all test conditions were maintained within the limits prescribed by the protocol and the test was considered valid.

Under the conditions of the present study 2,6-di-tert-butylphenol did not affect reproduction, survival or growth of Daphnia magna at an average measured concentration of 0.035 mg/L after 21 days of exposure (NOEC).