Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2017 to 02 October 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:

see below:-

Principles of method if other than guideline:

The total and heterotrophic respiration inhibition was evaluated using one set of vessels in which oxygen consumption was measured before and after amendment with a nitrification inhibitor (ATU), rather than with two discrete sets of vessels (i.e. with and without ATU) incubated for 3 hours as described in the OECD Guideline 209. This deviation is not considered to have adversely impacted the outcome of the study because it reduces the inherent variability within the test system and the validity criteria for the controls and reference substance were met for all respiration endpoints.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Purity: >98%; Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Yellow Liquid
- Storage: Room temperature (15 to 30°C)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Each treatment vessel contained a synthetic sewage concentrate, dechlorinated tap water, test or reference substance (blank controls had neither) and, for heterotrophic respiration assessment, a nitrification inhibitor to achieve total test volumes of 250 mL.

The test substance was weighed into individual foil boats and added directly to test vessels to achieve the respective nominal concentrations in the range-finding and definitive tests. Immediately after preparation, the pH of each vessel was determined and adjusted to within 7.5 ± 0.5 g/L with 40% NaOH, as necessary.

The reference substance, 3,5-dichlorophenol (3,5-DCP), was added the test vessels as a prepared aqueous stock solution (0.50 g/L) to achieve nominal concentrations of 0.1, 2.0 and 40 mg/L (single vessel for each) in the range-finding and definitive tests.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: sludge return line at Burley Menston sewage treatment works (West Yorkshire, UK), which has a predominantly domestic catchment.
- Method of cultivation: stored for 1 day before use; maintained at 20 ± 2°C and fed daily with synthetic sewage concentrate at a rate of 50 mL/L.
- Initial biomass concentration: 4.49 g/L (suspended solids concentration determined gravimetrically following homogenisation)
- Preparation of inoculum for exposure: adjusted with dechlorinated tap water to a nominal range of 3 ± 0.3 g/L. The pH was determined to be 7.28; no adjustment was required.
- Pretreatment: None.
- Test initiation: The test vessels for the definitive test were inoculated in seven batches of six at 30 minute intervals. Each prepared batch consisted of at one control vessel and either five test substance vessels or one three reference substance vessels and two further controls, as appropriate. Following inoculation, each vessel contained a nominal suspended solids concentration of ca.1.5 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Following oxygen consumption measurements to determine total respiration rates, a nitrification inhibitor (N-allylthiourea, ATU), was added as an aqueous stock solution (2.32 g/L) to the prepared blank control, test substance and reference substance vessels to give a final concentration of ca. 11.6 mg ATU/L in each. Oxygen consumption in the absence of nitrification was measured at least 10 minutes after amendment with ATU to assess heterotrophic respiration. Nitrification respiration was calculated as the difference between total respiration and heterotrophic respiration.

pH:

Ranged from 7.02 to 7.70 in definitive test

Nominal and measured concentrations:

Range-finding exposure: control, 1, 10, 100 and 1000 mg/L (nominal)
Definitive: control, 4, 12, 37, 111, 333 and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks (containing a total volume of 250 mL)
- No. of vessels per concentration (replicates):
> Test substance: 4, 12, 37, 111, 333 and 1000 mg/L (five replicates each; each test vessel was used as an individual replicate for respiration assessments)
> Reference substance: 0.1, 2.0 and 40 mg/L (single vessel for each)
- No. of vessels per control (replicates): Six, although the raw data indicate there were three additional vessels for which vessel preparation are incomplete; the data for these vessels were reported but not included in mean control calculations.
- No. of vessels per vehicle control (replicates): Not applicable
- No. of vessels per abiotic control (replicates): Not applicable
- Aeration: Yes; at a rate sufficient to keep the test samples adequately mixed
- Nutrients provided for bacteria: Pre-made synthetic sewage concentrate (SI093; Strathkelvin Instruments) purchased from Strathkelvin Instruments prepared in water (250 mL)
- Nitrification inhibitor used: N-allylthiourea (ATU)
- Biomass loading rate: Following inoculation, each vessel contained a nominal suspended solids concentration of ca.1.5 g/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Particulate matter: not specified

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted to within 7.5 ± 0.5 g/L with 40% NaOH, as necessary
- Photoperiod: Not specified
- Light intensity: Not specified
- Details on termination of incubation: At the end of the 3 hour incubation period, a portion (20 mL) of each test preparation was transferred to an appropriate sample tube containing a PTFE stirrer. The dissolved oxygen (DO) electrode was sealed in the neck of the flask, ensuring air was completely excluded. The flask contents were stirred at a constant rate during DO measurements. Oxygen consumption was measured over a period of up to 10 minutes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

48 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

67.7 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

4.7 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

28.8 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 4 mg/L

Nominal / measured:

nominal

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

The results of the range finding test indicated that the total, heterotrophic and nitrification respiration EC50 values for the test substance were between 10 and 100 mg/L; definitive test concentrations were selected accordingly.

Dissolved oxygen measurements were plotted over time. Respiration rates (mg/L/h) were determined from the gradient of the linear portion of the graph between 2.0 mg O2/L and 7.0 mg O2/L, where possible. In some cases, however, it was necessary to use readings below 2.0 mg O2/L; these were considered acceptable if the data used were with the linear portion of the curve and the rate remained consistent over the lower boundary. Graphical data and calculated respiration rates were generated using Strathkelvin Strathtox software.

Statistically significant effects on respiration of activated sludge were observed following a 3-hour exposure to the test substance.

The blank control respiration rate was ≥ 20 mg O2/g/h. The coefficient of variation of the blank control respiration rates were ≤ 30%.

Results with reference substance (positive control):

The 3-hour EC50 values for the reference item, 3, 5-dichlorophenol, were determined to be 3.1 mg/L (total respiration), 11.5 mg/L (heterotrophic respiration) and 1.1 mg/L (nitrification respiration).

Reported statistics and error estimates:

Statistical analysis was performed using the CETIS program v 1.8.6.8. Linear interpolation analysis was performed to estimate total, heterotrophic and nitrification respiration EC10, EC20 and EC50 values for the reference substance and test substance, where appropriate. Where possible, 95% confidence limits were calculated for the EC10, EC20 and EC50 values reported for the test substance. It was not possible to calculate 95 % confidence limits for the reference substance due to the limited data set.

Respiration Rates and Percent Inhibition for the Test Substance 

Nominal Test Substance Concentration (mg/L)	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate * (mg/L/h)	Nitrification Respiration Rate ** (mg/L/h)	Total Respiration Inhibition (%)	Heterotrophic Respiration Inhibition (%)	Nitrification Respiration Inhibition (%)
Control	46.8	22.2	24.6	-	-	-
4	45.8	23.0	22.8	2	-4	7
12	32.8	15.1	17.7	30	32	28
37#	26.0	15.3	10.7	44	31	57
111	15.0	8.1	6.9	68	63	72
333	5.7	4.8	0.9	88	78	96
1000	1.4	2.1	-0.6	97	91	103

 - Not Applicable

* Respiration in the presence of a nitrification inhibitor, N-allylthiourea (11.6 mg ATU/L).

** Calculated as the difference of total respiration rate and heterotrophic respiration rate.

# The mean values for the 37 mg/L group have been calculated using only 4 out of the 5 replicates because the nitrification value for replicate A was identified to be a statistical outlier.  

 

Respiration Rates and Percent Inhibition for the Reference Substance 

3,5-DCP Nominal Concentration (mg/L)	Total Respiration Rate (mg/L/h)	Heterotrophic Respiration Rate* (mg/L/h)	Nitrification Respiration Rate** (mg/L/h)	Total Respiration Inhibition (%)	Heterotrophic Respiration Inhibition (%)	Nitrification Respiration Inhibition (%)
0.1	60.2	28.3	31.9	-28.8	-27.7	-29.8
2.0	29.9	23.3	6.6	36.0	-5.1	73.0
40	4.2	3.7	0.5	91.0	83.3	98.0

Negative values indicate stimulated respiration.

* Respiration in the presence of a nitrification inhibitor, N-allylthiourea (11.6 mg ATU/L).

** Calculated as the difference of total respiration rate and heterotrophic respiration rate.

Validity criteria fulfilled:

yes

Conclusions:

Statistically significant effects on respiration of activated sludge were observed following a 3-hour exposure to the test substance. The 3-hour EC50 values for the test substance were determined to be 48.0 mg/L (total respiration), 67.7 mg/L (heterotrophic respiration) and 28.8 mg/L (nitrification respiration). The 3-hour EC10 values for the test substance were determined to be 5.6 mg/L (total respiration), 5.7 mg/L (heterotrophic respiration) and 4.7 mg/L (nitrification respiration).

Executive summary:

The study was conducted to assess the effect of the test substance on the activated sludge micro-organisms of the biological (aerobic) stage of waste-water treatment plants in accordance with OECD Test Guideline 209, under GLP conditions. The activated sludge inoculum was collected from the sludge return line at Burley Menston Sewage Treatment Works (West Yorkshire, U.K.), which has a predominantly domestic catchment.

 

Based on the results of a range finding test, a definitive test was conducted at the nominal test concentrations of 4, 12, 37, 111, 333 and 1000 mg/L. The test preparations were maintained at 20 ± 2°C with aeration for the 3-hour incubation period. At the end of the incubation period, oxygen consumption was measured over a period of up to 10 minutes. Following oxygen consumption measurements to determine total respiration rates, a nitrification inhibitor (N-allylthiourea, ATU), was added as an aqueous stock solution to the prepared blank control, test substance and reference substance vessels. Oxygen consumption in the absence of nitrification was measured at least 10 minutes after amendment with ATU to assess heterotrophic respiration. Nitrification respiration was calculated as the difference between total respiration and heterotrophic respiration.

 

Statistically significant effects on respiration of activated sludge were observed following a 3-hour exposure to the test substance. The 3-hour EC50 values for the test substance were determined to be 48.0 mg/L (total respiration), 67.7 mg/L (heterotrophic respiration) and 28.8 mg/L (nitrification respiration). The 3-hour EC10 values for the test substance were determined to be 5.6 mg/L (total respiration), 5.7 mg/L (heterotrophic respiration) and 4.7 mg/L (nitrification respiration).

 

The 3-hour EC50 values for the reference item, 3, 5-dichlorophenol, were determined to be 3.1 mg/L (total respiration), 11.5 mg/L (heterotrophic respiration) and 1.1 mg/L (nitrification respiration).

Description of key information

In an OECD Guideline 209 study, conducted according to GLP, the inhibition of total respiration was observed following a 3-hour exposure of activated sewage sludge to the test substance, resulting in an EC50 values of 48.0 mg/L (total respiration), 67.7 mg/L (heterotrophic respiration) and 28.8 mg/L (nitrification respiration). The 3-hour EC10 values for the test substance were determined to be 5.6 mg/L (total respiration), 5.7 mg/L (heterotrophic respiration) and 4.7 mg/L (nitrification respiration) (Smithers Viscient (ESG) Ltd, 2018).

Key value for chemical safety assessment

EC50 for microorganisms:

48 mg/L

EC10 or NOEC for microorganisms:

5.6 mg/L