Reaction mass of 2-(1,1-dimethylpropyl)anthraquinone and 2-(1,2-dimethylpropyl)anthraquinone

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March - 07 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”

Version / remarks:

2000-03-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

December 14, 2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: light sensitivity is expected

Analytical monitoring:

yes

Details on sampling:

- Sampling: Single 2 mL samples for analysis were taken from all test concentrations and the solvent-control at the beginning and at the end of three intervals of 48 hours (nominal days 0 and 2, 8 and 10, 14 and 16).
- Sample storage conditions before analysis: in freezer

Vehicle:

yes

Remarks:

DMSO

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:Test concentrations were prepared using an organic pre-solvent Dimethylsulphoxide (DMSO). Stock solutions in DMSO were prepared at a factor of 10,000 exceeding the required test concentrations.
Test medium (M7 medium) was spiked with the respective stock in DMSO at a volume of 100 μL per litre of medium and stirred for approximately 15-20 minutes before use. Due to the expected light sensitivity of the test item, preparation was performed under dimmed light conditions as much as possible.
- Controls: blank control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not indicated.
- Evidence of undissolved material: no, all final test solutions were clear and colourless

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: In-house laboratory culture with a known history
- Age of parental stock: young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
- 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.
- Feeding during test: Food type, frequency and amount: twice daily, an amount of 0.30 mg C/daphnia/day in a form of a mixture of Chlorella pyrenoidosa suspension combined with Marinure2 (TOC ratio of this mixture was 5:1). On weekend days, an amount of 0.60 mg C/daphnia/day was added in one single feed.

Breeding:
Start of each batch is with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks.
Renewal of the cultures: after 7 days of cultivation, half of the medium, twice a week.
Feeding: daily, a suspension of fresh water algae

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

180 mg/L as CaCO3

Test temperature:

19.7 - 20.6 ºC

pH:

7.1 - 8.2

Dissolved oxygen:

4.2 - 9.6 mg/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal: 18, 32, 56, 100 and 180 μg/L
Measured: 11, 21, 35, 67 and 131 µg/L calculated as the arithmetic mean of the geometric means of samples taken during three renewal intervals from freshly prepared 2-amylanthraquinone solutions (Ct=0, Ct=8 and Ct=14) and from the 48-hour old
solutions (Ct=2, Ct=10 and Ct=16).

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 ml (6 x Ø 3.5 cm) volume, all-glass covered with a Perspex plate.
- Aeration: not indicated
- Renewal rate of test solution: every 48 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Test medium: M7
- Source/preparation of dilution water: tap water purified by Reverse Osmosis
- Total organic carbon: 0.07 mg/L (measured at the start of the test, using a Shimadzu TOC-VCPH total organic carbon analyzer combined with a Shimadzu ASI-V auto sampler.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily
- Light intensity: at the start: 92-97 lux; at the end: 93-98 lux

EFFECT PARAMETERS MEASURED:
Parental daphnids
Condition: Every workday and upon renewal on non-workdays, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday and upon renewal on non-workdays.
Body length: At the end of the test.
Offspring
Appearance first brood: When observed.
Newborn daphnids: Every workday and upon renewal on non-workdays, the number of newborn young was counted and the condition of the young recorded. Thereafter, the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Two preliminary tests were performed prior to the reproduction test. A first test was stopped after one day due to the fact that the light intensity was too high and hence not reported. In the second test, each test group consisted of two replicates containing a total of ten daphnids (five each). The total test period was nine days. Test conditions were kept as similar as possible to those applied in the reproduction test including feeding. Test solutions were renewed on days 2, 5, 6 and 8 during the test. Samples for possible analysis were taken at days 0 (fresh), 2 (fresh and old), 5 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions without daphnia and algae.
- Test concentrations: 15, 50 and 150 μg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

84 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: mean measured value is calculated as the aritmethic mean of the 3 geometric means of the samples taken during the renewal intervals (new and old solutions)

Details on results:

Nominal versus measured:
During the range finding test, samples taken from nominal 150 μg/L were analysed. The results indicated that the concentration in the freshly prepared solutions were mostly in agreement with nominal (67-92%). The concentrations measured in the spent solutions decreased by 20%, 23% and 47% during exposure periods of 24, 48 and 72 hours, respectively. The concentrations measured in the solutions incubated without daphnia and algae remained stable within >90% of the initial measured concentrations. It was therefore concluded that 2-amylanthraquinone was not subject to any degradation. The decreasing concentrations in the medium were most likely related to adsorption of the test item to algae that were consequently consumed by the daphnia.
During the final test, the stability of the test solutions during the renewal periods decreased as exposure progressed. This decrease was assumed to be related to consumption and/or absorption of test item by the parental daphnids and the increasing numbers of young towards the end of the 21-day exposure period.

Parental mortality:
One out of ten daphnids exposed to the blank-control, the solvent-control, 11 and 131 μg/L died during the test period. The observed mortality was statistically insignificant. Hence, the NOEC for survival equalled the highest test group, being a concentration of 131 μg/L.

Age at first reproduction:
No delay in the appearance of the first brood was observed in any of the concentrations tested when compared to the pooled control.

Reproduction:
On average, 227 offspring were produced per surviving daphnid in the blank-control and 221 offspring in the solvent-control. Statistical analysis showed that the two controls were not statistically different from each other. Therefore, the controls were pooled for comparison with the treatment groups. Statistical analysis showed that the reproduction of the daphnids was statistically significantly reduced at concentrations of 35 μg/L and higher.

Body length:
The mean lengths recorded in the blank-control and solvent-control were 4.91 and 4.75 mm, respectively. The two controls were statistically different from each other and the solvent-control was thus selected for comparison with the treatment groups.
The mean length of the parents exposed to 11 and 21 μg/L were comparable to the solvent-control, while the lengths of parents exposed to higher concentrations were increasingly reduced with a maximum of 13.5% at the highest test concentration. Statistically significant reduction of growth was found at test item concentrations of 35 μg/L and higher. The EC10 for effects on growth was determined to be 88 μg/L.

Effect parameters as stated in the study report:
Parameter Concentration (µg/L)
NOEC for reproduction 21
EC10 for reproduction 84 (95% CI 27-260)
EC50 for reproduction >131
NOEC for mortality 131
NOEC for growth 21
EC10 for growth 88 (95% CI 69-112)

Reported statistics and error estimates:

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).
The following statistical procedures were used to determine the NOEC for reproduction and growth:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
- Differences between control groups: Student-t-test Procedure
- Differences between treatments and the control: Reproduction: Step-down Jonckheere-Terpstra test Procedure; Length: Williams Multiple Sequential t-test Procedure
Mortality of parental daphnids was analysed with the Fisher`s Exact Binomial Test with Bonferroni Correction.

EC-values for reproduction and length:
The EC10 was determined using the 3-parameter Normal CDF (Cumulative Distribution function) with cumulative offspring per surviving parent at day 21. This program uses regression analyses and is used for non-linear responses.
No EC50 values could be calculated as the observed effects were lower than 50%.

Validity criteria fulfilled:

yes

Remarks:

see "overall remarks"

Conclusions:

The EC10 for reproduction is 84 µg/L based on mean measured concentrations.

Executive summary:

The long-term toxicity of the test item to aquatic invertebrates was studied in a daphnia magna reproduction study according to OECD 211 and under GLP conditions.

10 neonates (<24 h old) were individually exposed in a semi-static sytem to nominal test concentrations of 18, 32, 56, 100 and 180 µg/L for 21 days with test solutions renewed every 48 hours. Additionally, a blank-control and solvent-control were included. Parental mortality, number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Samples taken at the beginning and the end of three 48-hour renewal intervals were analyzed. The concentrations measured in the freshly prepared solutions were in agreement with nominal (86 - 104% of nominal). Measured concentrations decreased only slightly during the first renewal period (day 0-2), but the decreases were significant during the second and third analyzed renewal periods (day 8-10 and day 14-16). Mean measured concentrations were calculated as arithmetic mean of the 3 geometric mean measured concentrations of the 3 analyzed renewal periods and were 11, 21, 35, 67 and 131 μg/L for nominal concentrations of 18, 32, 56, 100 and 180 µg/L, respectively. Mortality in the controls and the test concentrations did not exceed 10%. Reproduction was statistically significantly reduced at test concentrations of 35 µg/L and above when compared to the pooled control group. The onset of reproduction was not delayed in any of the test concentrations when compared to the controls. The mean lengths of parents exposed to 35 µg/L and higher were statistically significantly reduced as compared to the solvent-control.

The overall effect parameter of the study is the EC10 for reproduction of 84 µg/L. The study met de validity criteria and is considered reliable without restrictions.

Description of key information

The long-term toxicity of the test item to aquatic invertebrates was studied in a daphnia magna reproduction study according to OECD 211 and under GLP conditions.

10 neonates (<24 h old) were individually exposed in a semi-static sytem to nominal test concentrations of 18, 32, 56, 100 and 180 µg/L for 21 days with test solutions renewed every 48 hours. Additionally, a blank-control and solvent-control were included. Parental mortality, number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded and the lengths of the surviving parental daphnids were measured at the end of the test.

Samples taken at the beginning and the end of three 48-hour renewal intervals were analyzed. The concentrations measured in the freshly prepared solutions were in agreement with nominal (86 - 104% of nominal). Measured concentrations decreased only slightly during the first renewal period (day 0-2), but the decreases were significant during the second and third analyzed renewal periods (day 8-10 and day 14-16). Mean measured concentrations were calculated as arithmetic mean of the 3 geometric mean measured concentrations of the 3 analyzed renewal periods and were 11, 21, 35, 67 and 131 μg/L for nominal concentrations of 18, 32, 56, 100 and 180 µg/L, respectively. Mortality in the controls and the test concentrations did not exceed 10%. Reproduction was statistically significantly reduced at test concentrations of 35 µg/L and above when compared to the pooled control group. The onset of reproduction was not delayed in any of the test concentrations when compared to the controls. The mean lengths of parents exposed to 35 µg/L and higher were statistically significantly reduced as compared to the solvent-control.

The overall effect parameter of the study is the EC10 for reproduction of 84 µg/L. The study met de validity criteria and is considered reliable without restrictions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

84 µg/L