2,5-di-tert-pentylhydroquinone

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:

30 September 2016 to 21 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

OECD guidelines for Testing of Chemicals, guideline No. 211: "Daphnia magna, Reproduction Test", Adopted: October 2012.

Deviations:

yes

Remarks:

See "Any other information" for details.

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

Council Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.20. "Daphnia magna Reproduction Test".

Deviations:

yes

Remarks:

See "Any other information" for details.

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Remarks:

UPLC-MS

Details on sampling:

Single samples for analysis were taken from all test concentrations and the solvent-control.
Sampling:
Frequency: One day before the start of the test (to check the test system functioning), at the start and after 7, 14 and 21 days of exposure.
Volume: 2.0 mL
Storage: Samples not analysed on the day of sampling were stored in a freezer (≤ -15°C) until analysis.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Stability and concentration of the stock solutions used for dosing the test concentrations was also verified with samples taken from freshly prepared and two- and three-day old stock solutions on days 1, 4 and 6 (sample volume 0.5 mL). These samples were stored in a freezer (≤ -15°C) until analysis.

Vehicle:

yes

Remarks:

Dimethyl Formamide

Details on test solutions:

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.1% and the item was poorly soluble in test medium. No correction was made for the purity/composition of the test substance.

Range-finding test (semi-static)
Preparation of test solutions for the semi-static range-finding test started with a concentration of 1.0 mg/L that was magnetically stirred for three days to achieve maximum dissolution. The resulting dispersion was filtered through a 0.45 μm membrane filter (Whatman RC55) to remove the fraction of undissolved test item. The clear and colourless Saturated Solution (SS) was used as highest test concentration and used to prepare two lower test concentrations (1.0 and 10% SS) by serial dilution in test medium.

Final test (flow-through)
Stock solutions were prepared in Dimethyl Formamide (DMF; Merck, Darmstadt, Germany, ≥ 99.8%) at 2 or 3-day 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 DMF resulting in clear and colourless solutions.
Exact volumes of the respective stock solutions and the dilution water (M7-medium with algae suspension) were dosed using diluters (test item stocks) or peristaltic pumps (M7 dilution water) into the various mixing vessels. The dosed volumes of test item stock and the M7-medium were continuously stirred in a mixing vessel. From this vessel, the mixture was transferred into stainless steel test vessels containing the daphnia. The renewal rate of the test solutions was ~700 mL/hour. The whole system was checked daily.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical 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.

Breeding
Start of each batch: With newborn daphnids, i.e. less than 3 days old by placing about 250 of them into 5 litres of 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.
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 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

Remarks on exposure duration:

As per the test guideline.

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Total hardness varied between 179 and 196 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the study plan (>140 mg CaCO3 per liter).

Test temperature:

The temperatures in the test media varied between 19.9 and 20.9°C. The temperature continuously measured in one of the control vessels varied between 19 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

pH:

The pH remained within the range of 7.3 to 8.3 throughout the test and was thus maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).

Dissolved oxygen:

The oxygen concentration in all test solutions generally remained within the range of 6.0 to 9.7 mg/L during the 21-day exposure period, except for day 7 when oxygen concentrations had dropped to values between 2.4 and 4.2 in the solutions dosed with DMF. It was decided to aerate the solutions from that day onwards and oxygen quickly recovered to normal values, which were maintained for the rest of the study period.

Salinity:

Not applicable.

Conductivity:

Not specified

Nominal and measured concentrations:

Analyses showed that actual measured concentrations were at approximately 70 to 80% relative to the targets at the start of the test. Measured concentrations decreased only slightly during the first 14 days at the target concentrations of 18 to 72 μg/L, whereas at the lower target concentrations already a slightly higher decrease was observed during this test period.
Results of the samples taken at the end of the test showed that measured concentrations had decreased rather drastic and, again, with the highest relative decreases at the lowest concentrations. The flow-through system functioned without any flaws during the whole test period and thus no mechanistic issue had occurred. The most plausible reason for this decrease, which was already visible after 7 and 14 days, was adsorption and or consumption of test item to the increasing biomass of organic material in the test system. The organic material being offspring that was mainly produced during the 2nd half of the study period, algae that adsorbed to the tubing and walls of the test system and also the possible presence of bacteria growing against the walls of the system. It was consequently decided to calculate the mean exposure concentrations from all four analyses occasions, which resulted in a worstcase scenario for the actual exposure concentrations. Theoretically, daphnids might have been exposed to concentrations close to the target during the whole test period but then, towards the study end, partly by i.e. indigestion of test item adsorbed to or by the edible sources present in the system (algae, bacteria).
The range tested based on average measured concentrations corresponded to 2.1, 4.5, 11, 24 and 48 μg/L.

Details on test conditions:

Range-finding test (semi-static)
A preliminary test was performed prior to the definitive reproduction test. The concentrations of Lowinox® AH25 tested corresponded to 1.0, 10 and 100% of a 0.45 μm filtered solution that was prepared at a loading rate of 1.0 mg/L. A control group was also included. Each concentration consisted of two replicates containing a total of ten daphnids (five each). The total test period was ten days. Test conditions were held as similar as possible to those applied in the reproduction test including feeding. Test solutions were renewed on days 1, 3 and 6 during the test. Samples for possible analysis were taken at days 0 (fresh), 1 (fresh and old), 3 (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, but without daphnia and algae.

Solubility test using daphnia test medium
After the range-finding test, a solubility test was performed to confirm the expectation that the solubility in test medium was much lower than the solubility in pure water that was indicated to be 1 g/L at 20°C. The column elution method was used to determine the medium solubility.

Reproduction test (Flow-through)
Test concentrations
Lowinox® AH25: Target concentrations of 4.5, 9.0, 18, 36 and 72 μg/L
Controls: Test medium without test item or other additives and one control containing test medium with the additive (DMF) used in the treatment of the stock solutions.

Test procedure and 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: At the start of the experiment (nominal day 0) 20 neonate daphnids, less than one day old, per group were introduced to 4 stainless steel meshes (5 per mesh).
Light: 16 h photoperiod daily; intensity at the start: 679-796 lux; intensity at the end: 624-744 lux
Feeding: A Chlorella sp. suspension was fed continuously. The daily ration theoretically corresponded to 0.4 mg C/daphnia/day. The daily ration provided was higher than the recommended value for daily feeding per daphnid in the reproduction test according to the OECD
Guideline 211 (0.1-0.2 mg C/daphnia/day), to correct for the waste of algae that was inherent to the use of a flow-through design.

Measurements and recordings
Parental daphnids
Condition: The number of living and dead parental daphnids was daily recorded. Dead daphnids were removed when observed.
Presence of eggs in: Daily
Body length: At the end of the test.
Offspring
Appearance first brood: When observed.
Newborn daphnids: Daily the number of neonates was counted and the condition of the neonates recorded. Thereafter, the neonates were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.
Test medium
Temperature, oxygen and pH: At the start of the test and weekly thereafter.
Hardness: At the start of the test and weekly thereafter in the controls and the highest test concentration.
Light: At the start and the end of the test.

Interpretation
Data handling
The values for reproduction observed at the various concentrations of the test item were expressed as mean number of living neonates per introduced parent in-line with the recommendations of OECD guidelines number 211. The mean values for reproduction at each concentration were compared to those recorded in the control group(s) on the various days of recording. Further, the lengths of the parental daphnids (day 21) exposed to the test item were compared to the control group(s).
Exposure concentrations
The exposure concentrations were calculated as the (arithmetic) mean of the concentrations measured at the start, after 7, 14 and 21 days of exposure.
Statistical analysis
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 treatments and the (pooled) control: Williams Multiple (Sequential) t-test Procedure
Parental survival was not statistically analysed, as the mortality did not exceed 10%.

EC-values for reproduction:
No EC10 / EC50 values could be calculated as the observed effects were lower than 10% / 50%.
All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Acceptability of the test
1. The mortality of the parent animals (female Daphnia) in the control did not exceed 20% at the end of the test (both controls; 0%).
2. The average cumulative number of young per female in the controls after 21 days was ≥ 60 (Blank-control: 238, Solvent-control: 224).

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

48 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction & mortality

Remarks on result:

other: This concentration was expected to be at or close to the maximum soluble concentration of Lowinox® AH25 in test medium.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

Range-finding test
One of the ten daphnids introduced to the control treatment did not survive the exposure.
Three daphnids exposed to 1.0% SS did not survive the test period and one daphnid did not survive at 10% SS. Nine of the ten daphnids exposed to the undiluted (100%) SS did not survive the test period.
First eggs in the brood pouch were recorded on day 7 of exposure and the first offspring was observed on day 9 in the control and all solutions up to and including 10% SS. No offspring was observed in the 100% SS.
Based on these results it was concluded that no effects on survival or reproduction of daphnia were to be expected when exposed up to concentrations present in a solution containing 10% SS.
Analyses showed that the measured concentrations in the freshly prepared SS ranged between 260 and 392 μg/L. The concentrations measured in the dilutions (1.0 and 10% SS) were more or less in agreement with the dilution factor. The concentrations decreased rapidly during the exposure period with the highest decreases observed towards the lowest test groups. It was further observed that the decreases were slightly lower in the solutions incubated without algae and daphnia when compared to the solutions with daphnia and algae.
It was consequently decided that a flow-through system would be the most suitable test system for the definitive test in order to maintain stable exposure concentrations.
Test conditions during the range-finding test were maintained within the limits prescribed by the study plan.

Solubility test using daphnia test medium
After the range-finding test, a medium solubility test was performed. This was based on the fact that the solubility in test medium was expected to be much lower than the solubility in pure water (1 g/L at 20 °C). Formulation during the range-finding test showed that a concentration of 1.0 mg/L could not be dissolved. Samples taken from the SS prepared at 1.0 mg/L during the range-finding test showed a mean measured concentration of ~ 300 μg/L.
The solubility test should further confirm whether the low measured concentration in the range-finding test actually represented the maximum soluble concentration in test medium.
The solubility at 20 °C in daphnia test medium (M7) was 64.8 μg/L, which confirmed the expectation that the solubility in test medium was much lower than in purified water.
Hence, the measured concentrations of ~ 300 μg/L in the SS freshly prepared at 1.0 mg/L still exceeded the actual limit of solubility in test medium. It was therefore concluded that the effects as observed in the range-finding test at this concentration might be related to testing above the solubility limit. It was consequently decided to test up to a maximum concentration of 72 μg/L in the definitive test as not to exceed the limit of solubility by too much.

Reproduction test
Measured concentrations
Analyses of samples taken from the freshly prepared and 48- or 72-hour old stock solutions showed that the stocks in DMF were prepared correctly and that they were stable for at least 72 hours, i.e. the maximum period before renewal of the stocks in the flow-through system.
Analyses showed that actual measured concentrations were at approximately 70 to 80% relative to the targets at the start of the test. Measured concentrations decreased only slightly during the first 14 days at the target concentrations of 18 to 72 μg/L, whereas at the lower target concentrations already a slightly higher decrease was observed during this test period.
Results of the samples taken at the end of the test showed that measured concentrations had decreased rather drastic and, again, with the highest relative decreases at the lowest concentrations. The flow-through system functioned without any flaws during the whole test period and thus no mechanistic issue had occurred. The most plausible reason for this decrease, which was already visible after 7 and 14 days, was adsorption and or consumption of test item to the increasing biomass of organic material in the test system. The organic material being offspring that was mainly produced during the 2nd half of the study period, algae that adsorbed to the tubing and walls of the test system and also the possible presence of bacteria growing against the walls of the system. It was consequently decided to calculate the mean exposure concentrations from all four analyses occasions, which resulted in a worst case scenario for the actual exposure concentrations. Theoretically, daphnids might have been exposed to concentrations close to the target during the whole test period but then, towards the study end, partly by i.e. indigestion of test item adsorbed to or by the edible sources present in the system (algae, bacteria).
The range tested based on average measured concentrations corresponded to 2.1, 4.5, 11, 24 and 48 μg/L.

Condition of parental daphnids
All parental daphnids survived the test period in both the solvent- and blank-control. Survival in the various test item treated groups was also high. One parent died in test groups containing average concentrations of 2.1 and 11 μg/L. Hence, no treatment related mortality of parental daphnids was observed and the NOEC for survival was consequently set at the highest tested concentration, being 48 μg/L.

Age at first reproduction
The first brood was observed on day 7 in each of the mesh containers of each tested group. The NOEC for age at first reproduction equalled the highest test group of 48 μg/L.

Reproduction
On average, 238 and 224 offspring were produced per surviving daphnid in the blank-control and solvent-control, respectively. Statistical analysis showed no difference between the two controls and therefore controls were pooled for comparison with the test item treatments.
The average number of offspring in the treatment groups were 234, 234, 268, 253 and 242 at 2.1, 4.5, 11, 24 and 48 μg/L, respectively. Reproduction in all treatments was thus comparable to the reproduction in the control groups. Statistical analysis confirmed that the reproduction of the daphnids was not statistically significantly reduced at any of the concentrations tested. Hence, the NOEC for reproduction was 48 μg/L.
There were only a few recordings of immobile young or appearance of unhatched (aborted) eggs during the 21-day test period. These recordings were not treatment related.

Body length
The mean length of the daphnids at the end of the test were 4.43 and 4.41 mm for the blank control and solvent-control, respectively. Statistical analysis showed no difference between the two controls and therefore controls were pooled for comparison with the test item treatments.
The mean length of the daphnids in the various treatment groups were 4.47, 4.31, 4.45, 4.22 and 4.29 mm, respectively. Statistical analysis showed that there was a statistically significant reduction of growth in the two highest test concentrations when compared to the pooled control group. Hence, the NOEC for growth was set at 11 μg/L.

Experimental conditions
The pH remained within the range of 7.3 to 8.3 throughout the test and was thus maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).
The oxygen concentration in all test solutions generally remained within the range of 6.0 to 9.7 mg/L during the 21-day exposure period, except for day 7 when oxygen concentrations had dropped to values between 2.4 and 4.2 in the solutions dosed with DMF. It was decided to aerate the solutions from that day onwards and oxygen quickly recovered to normal values, which were maintained for the rest of the study period.
The temperatures in the test media varied between 19.9 and 20.9 °C. The temperature continuously measured in one of the control vessels varied between 19 and 21 °C during the test, and complied with the requirements as laid down in the study plan (18-22 °C, constant within 2 °C).
Total hardness varied between 179 and 196 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the study plan (>140 mg CaCO3 per liter).
The total dissolved organic carbon content of the M7 medium was 0.26 mg/L. This complied with the requirements as laid down in the guidelines (TOC < 2 mg/L).

Results with reference substance (positive control):

Not specified.

Reproduction and mortality of the adult daphnids in the range-finding test

Day	Lowinox® AH25, % SS prepared at 1.0 mg/L
	Control		1.0		10		100
	A	B	A	B	A	B	A	B
8 9 10	- 11 -	- 8 -	- - 11	- 7 -	- 3 -	- - 12	- - -	- - -
Total Average per group	11	8 10	11	7 9	3	12 8	-	- -
Number of survived parental daphnids	4	5	3	4	5	4	0	1

 

Target versus measured exposure concentrations

Lowinox® AH25 Concentration target (μg/L)	Measured concentration (μg/L)				Average exposure concentration (μg/L)*
	Day 0	Day 7	Day 14	Day 21
4.5 9.0 18 36 72	3.51 6.54 15.0 29.4 59.8	2.86 5.89 13.9 29.1 60.6	1.82 4.59 12.4 25.9 55.1	0.13 1.00 2.08 11.4 16.2	2.1 (47) 4.5 (50) 11 (61) 24 (67) 48 (67)

* Between brackets: percentage recovery relative to the target

 

Mortality (immobility) of parental daphnids at the end of the test

Lowinox® AH25 Average Concentration (μg/L)	Introduced	Mobile	Immobile	% Immobility
Blank-control Solvent-control 2.1 4.5 11 24 48	10 10 10 10 10 10 10	10 10 9 10 9 10 10	0 0 1 0 1 0 0	0 0 10 0 10 0 0

 

Age of first reproduction (day)

Lowinox® AH25 Average Concentration (μg/L)	Mean	Std. Dev.	n	% increase
Pooled-control 2.1 4.5 11 24 48	7.5 7.5 7.5 7.5 7.5 7.5	0.00 0.00 0.00 0.00 0.00 0.00	8 4 4 4 4 4	0.0 0.0 0.0 0.0 0.0

n – number of replicate mesh containers with reproductive daphnids

 

Group mean cumulative number of juveniles per introduced parent and reduction of reproduction at the end of the test

Lowinox® AH25 Average Concentration (μg/L)	Mean	Std. Dev.	n	% Reduction
Pooled Control 2.1 4.5 11 24 48	230.9 234.1 233.9 268.4 252.5 242.1	15.93 28.62 33.29 33.28 28.39 45.26	8 4 4 4 4 4	-1.4 -1.3 -16.2 -9.3 -4.8

 

Incidence of immobilised offspring and aborted eggs

Abnormality	Lowinox® AH25, Concentration (μg/L)
	Blank-control	Solvent-control	2.1	4.5	11	24	48
Immobile offspring Aborted eggs	0 0	27 1	0 0	73 0	4 11	9 8	13 11

 

Group mean body lengths (mm) and reduction of growth of parental daphnids at the end of the test

Lowinox® AH25 Average Concentration (μg/L)	Mean (mm)	Std. Dev.	n	% Reduction
Pooled Control 2.1 4.5 11 24 48	4.42 4.47 4.31 4.45 4.22 4.29	0.099 0.065 0.076 0.045 0.142 0.033	8 4 4 4 4 4	-1.3 2.5 -0.6 4.6 2.9

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the flow-through study, Lowinox® AH25 did not affect survival or reproduction of Daphnia magna at an average measured concentration of 48 μg/L after 21 days of exposure (NOEC). This concentration was expected to be at or close to the maximum soluble concentration of Lowinox® AH25 in test medium. For the additional parameter growth, a NOEC of 11 μg/L was obtained.

Executive summary:

Daphnia magna, 21-day reproduction study with Lowinox® AH25.

 

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 Council Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

 

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.1% and the item was poorly soluble in test medium.

 

A definitive reproduction test was performed in a flow-through system based on the result obtained in a preceding 10-day semi-static range-finding test.

 

Twenty neonate daphnids (<24h old), divided over four replicate mesh containers (five dapnids/mesh), were exposed per test group. The target concentrations of Lowinox® AH25 tested were 4.5, 9.0, 18, 36 and 72 μg/L. A blank- and solvent-control were included.

The study duration was 21 days and the test solutions were continuously renewed. The daphnids were fed with a Chlorella pyrenoidosa suspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was 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, after 7, 14 and 21 days of exposure. Analyses showed that actual measured concentrations were at approximately 70 to 80% relative to the targets at the start of the test. Measured concentrations decreased only slightly during the first 14 days at the target concentrations of 18 to 72 μg/L, whereas at the lower target concentrations already a slightly higher decrease was observed during this test period. Measured concentrations at the end of the test had decreased more significantly with the highest relative decreases at the lowest concentrations. The range tested based on average measured concentrations corresponded to 2.1, 4.5, 11, 24 and 48 μg/L.

 

All parental daphnids survived the test period in both the solvent- and blank-control. Survival in the various test item treated groups was also high. One parent died in test groups containing average concentrations of 2.1 and 11 μg/L. No parents died in the other test groups.

 

The average cumulative number of offspring per female were 238 and 224 in the blank- and solvent-control, respectively. In the treatment groups, the average number of offspring were 234, 234, 268, 253 and 242 at 2.1, 4.5, 11, 24 and 48 μg/L, respectively.

 

There was no delay in reproduction and there were no significant treatment related numbers of aborted eggs or immobile young at any of the concentrations tested.

 

The mean length of the parent daphnids in the two highest groups were reduced by 4.6 and 2.9% compared to the length of the daphnids exposed to the pooled control. The reduction in these two groups proved to be statistically significant. No, or no significant, reduction of length were observed in the lower test groups.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The effect parameters (μg/L) obtained in this study are summarized in the table below.

Parameter	Average measured concentration (μg/L)
NOEC for reproduction EC10,50for reproduction NOEC for mortality NOEC for growth	48 >48 48 11

 

It was concluded that under the conditions of the present flow-through study, Lowinox® AH25 did not affect survival or reproduction of Daphnia magna at an average measured concentration of 48 μg/L after 21 days of exposure (NOEC). This concentration was expected to be at or close to the maximum soluble concentration of Lowinox® AH25 in test medium.

For the additional parameter growth, a NOEC of 11 μg/L was obtained.

Description of key information

Lowinox® AH25 did not affect survival or reproduction of Daphnia magna at an average measured concentration of 48 μg/L after 21 days of exposure (NOEC). This concentration was expected to be at or close to the maximum soluble concentration of Lowinox® AH25 in test medium.

For the additional parameter growth, a NOEC of 11 μg/L was obtained.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

11 µg/L

Additional information

Daphnia magna, 21-day reproduction study with Lowinox® AH25. 

The batch of Lowinox® AH25 tested was a white to cream coloured powder with a purity of 95.1% and the item was poorly soluble in test medium.

 

A definitive reproduction test was performed in a flow-through system based on the result obtained in a preceding 10-day semi-static range-finding test.

Twenty neonate daphnids (<24h old), divided over four replicate mesh containers (five dapnids/mesh), were exposed per test group. The target concentrations of Lowinox® AH25 tested were 4.5, 9.0, 18, 36 and 72 μg/L. A blank- and solvent-control were included.

The study duration was 21 days and the test solutions were continuously renewed. The daphnids were fed with a Chlorella pyrenoidosa suspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the lengths of the surviving parental daphnids were measured.

 

All parental daphnids survived the test period in both the solvent- and blank-control. Survival in the various test item treated groups was also high. One parent died in test groups containing average concentrations of 2.1 and 11 μg/L. No parents died in the other test groups.

The average cumulative number of offspring per female were 238 and 224 in the blank- and solvent-control, respectively. In the treatment groups, the average number of offspring were 234, 234, 268, 253 and 242 at 2.1, 4.5, 11, 24 and 48 μg/L, respectively.

There was no delay in reproduction and there were no significant treatment related numbers of aborted eggs or immobile young at any of the concentrations tested.

 

The mean length of the parent daphnids in the two highest groups were reduced by 4.6 and 2.9% compared to the length of the daphnids exposed to the pooled control. The reduction in these two groups proved to be statistically significant. No, or no significant, reduction of length were observed in the lower test groups.

  

It was concluded that under the conditions of the present flow-through study, Lowinox® AH25 did not affect survival or reproduction of Daphnia magna at an average measured concentration of 48 μg/L after 21 days of exposure (NOEC). This concentration was expected to be at or close to the maximum soluble concentration of Lowinox® AH25 in test medium.

For the additional parameter growth, a NOEC of 11 μg/L was obtained.