5-bromo-5-nitro-1,3-dioxane

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Nov 2018 to 14 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 5-Bromo-5-nitro-1,3-dioxane
Batch (Lot) Number: 201807021
Expiry date: 30 Jun 2020 (expiry date) (taken from label)
Physical Description: White crystal powder
Storage Conditions: At room temperature

Analytical monitoring:

not specified

Details on sampling:

For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility.

Vehicle:

not specified

Details on test solutions:

Test Item – Preparation and Test Concentrations
The batch of 5-Bromo-5-nitro-1,3-dioxane tested was a white crystal powder with a purity of 99.63% (HPLC). No correction was made for the purity/composition of the test item. Preparation was as much as possible performed under or low light conditions.
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the amber coloured test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations (see Table 1 and Table 2). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded.

Reference Item – Preparation and Test Concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol.
A 1.0 g/L stock solution was prepared by dissolving 250.5 mg 3,5-dichlorophenol in Milli RO water and making up to a total volume of 250 mL. The pH as used for the test was 7.8 in the final test. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15℃) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium, resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.

Test organisms (species):

activated sludge

Details on inoculum:

Test system
Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas',
s -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
Preparation of the sludge The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/L, as used for the test. The pH was 7.7 one day before testing (See deviation in Appendix 4). The batch of sludge was used one day after collection. Therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Medium: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Test type:

not specified

Water media type:

saltwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 20 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:

The pH in all test vessels, before addition of sludge, was between 7.4 and 7.5. After the 3 hour exposure period the pH was between 8.0 and 8.2.

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C).

Salinity:

CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Nominal and measured concentrations:

The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 3.0 to 100 mg/L. Five replicates per concentration and six replicates for an untreated control group were tested.

Details on test conditions:

Contact time 3 hours, during which aeration and stirring took place.
Vessels All glass open bottles/vessels.
Milli-RO / Milli-RO water Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium 16 g peptone
(=sewage feed) 11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre
and filtered. The pH was within 7.5 ± 0.5.
Inhibitor of nitrification A 2.32 g/L solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 ml of this solution was added to 500 ml final test medium (final ATU concentration: 11.6 mg/L).
Air supply Clean, oil-free air.
Aeration The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
Test set up See paragraph 4.7.2
Performance of the test
The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter, 250 mL activated sludge was added. This was the start of the test. After the 3 hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Oxygen recording: Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 3.7 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 6 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 15 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Combined Limit/Range-Finding Test
The combined limit/range-finding showed 8%, 94% and 99% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50 was at a concentration between 10 and 100 mg/L.
There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Since there was no distorted dose response curve the full test will be based on only organic carbon oxidation.
There was no significant oxygen uptake from abiotic processes.
The temperature continuously measured in the temperature control vessels ranged between 19.3 and 23.5°C during the test, and was outside the range prescribed by the study plan (20 ± 2°C).
All test conditions, except the temperature and acceptability criteria prescribed by the study plan were met.

Final Test
Inhibition of the Respiration Rate
The effects observed were in agreement with what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and ECx values.
No statistically significant inhibition of the respiration rate of the sludge was recorded at 3.0 mg 5-Bromo-5-nitro-1,3-dioxane per liter (for more information on statistics see Appendix 4). At higher concentrations the inhibitory effect of 5-Bromo-5-nitro-1,3-dioxane on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 20% inhibition at 6.0 mg/L to 93% at 100 mg/L.

Final Test – Overview of Results
Treatment Concentration
(mg/L) Mean respiration rate % Inhibition of the respiration rate (mean value)
(mg O2/L h) (mg O2/g h)¹
Control 0 37.52 25.02
T1 3.0 34.66 23.11 7.64
T2 6.0 30.15 20.10 19.64*
T3 16 21.18 14.12 43.56*
T4 40 3.66 2.44 90.25*
T5 100 2.68 1.79 92.85*
¹) The amount of suspended solids in the final test mixture was 1.5 g/L.
* Statistically significantly different compared to control.

Results with reference substance (positive control):

Reference Item - Point Estimates From the 3-Param. Logistic CDF

Parameter EC50
Value [mg/L] 7.9
lower 95%-cl 7.5
upper 95%-cl 8.3

Reported statistics and error estimates:

ECx
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item, calculation of the ECx value was based on probit analysis using linear maximum likelihood regression, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).
Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test 5-Bromo-5-nitro-1,3-dioxane was not toxic to waste water bacteria (activated sludge) at a concentration of 3.0 mg/L (NOEC).
The EC10 was 3.7 mg/L (95% confidence interval: 2.6- 5.4 mg/L).
The EC20 was 6.0 mg/L (95% confidence interval: 4.5 – 8.0 mg/L).
The EC50 was 15 mg/L (95% confidence interval: 13 - 18 mg/L).

Executive summary:

The objective of the study was to evaluate 5-Bromo-5-nitro-1,3-dioxane for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC₅₀and/or the no - observed effect concentration (NOEC).

The influence of 5-Bromo-5-nitro-1,3-dioxane on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based onthe OECD guideline No. 209, 2010.

The batch of 5-Bromo-5-nitro-1,3-dioxane tested was a white crystal powder with a purity of 99.63% (HPLC). No correction was made for the purity/composition of the test item. Preparation was as much as possible performed under low light conditions.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli - RO water were added, resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 3.0 to 100 mg/L. Five replicates per concentration and six replicates for an untreated control group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at 3.0 mg 5-Bromo-5-nitro-1,3-dioxane per liter (for more information on statistics see Appendix 4). At higher concentrations the inhibitory effect of 5-Bromo-5-nitro-1,3-dioxane on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 20% inhibition at 6.0 mg/L to 93% at 400 mg/L.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

 

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

5-Bromo-5-nitro-1,3-dioxane was not toxic to waste water (activated sludge) bacteria at a concentration of 3.0 mg/L (NOEC).

The effect parameters for the inhibition of the respiration rate are presented below.

 (  ) Between brackets the 95% confidence intervals are given.

Parameter	5-Bromo-5-nitro-1,3-dioxane Concentration (mg/L)
NOEC	3.0
EC10	3.7 (2.6 – 5.4)
EC20	6.0 (4.5 – 8.0)
EC50	15 (13 - 18))

Description of key information

Under the conditions of the study, the test substance was not toxic to waste water bacteria (activated sludge) at a concentration of 3.0 mg/L (NOEC).

The EC10 was 3.7 mg/L (95% confidence interval: 2.6- 5.4 mg/L).

The EC20 was 6.0 mg/L (95% confidence interval: 4.5 – 8.0 mg/L).

The EC50 was 15 mg/L (95% confidence interval: 13 - 18 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

15 mg/L

EC10 or NOEC for microorganisms:

3.7 mg/L