Reaction mass of [[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid and Phosphonic acid, P-[(tetrahydro-2-hydroxy-2-oxido-4H-1,4,2-oxazaphosphorin-4-yl)methyl]-

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 28 June 2012; Experimental Completion Date: 16 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations:
10, 100 and 1000 mg active ingredient (ai)/L

- Sampling method:
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

RANGE-FINDING TEST TO MEASURE TOTAL RESPIRATION:
For this range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg active ingredient (ai)/L. The test item was dissolved directly in water. The test item contains 10.95% water therefore all test concentrations were corrected for this. In order to homogenize the sample the test item was warmed to approximately 50°C prior to weighing.

An amount of test item (2807 mg) was dissolved in water and the volume adjusted to 1 litre to give a 2500 mg ai/L stock solution from which dilutions were made to give 250 and 25 mg ai/L stock solutions. An aliquot (200 mL) of the 25 mg ai/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg ai/L. Similarly, aliquots (200 mL) of the 250 mg ai/L and 2500 mg ai/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg ai/L. The 1000 mg ai/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

The pH of the test item stock solutions were measured using a a WTW pH/Oxi 340I pH and dissolved oxygen meter (see Table 1 - attached background material) and adjusted to between pH 7.0 to pH 8.0. The control group was maintained under identical conditions but not exposed to the test item.

RANGE-FINDING TEST TO MEASURE HETEROTROPHIC RESPIRATION:
For this range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg active ingredient (ai)/L. The test item was dissolved directly in water. The test item contains 10.95% water therefore all test concentrations were corrected for this. In order to homogenize the sample the test item was warmed to approximately 50°C prior to weighing.

An amount of test item (2807 mg) was dissolved in water and the volume adjusted to 1 liter to give a 2500 mg ai/L stock solution from which dilutions were made to give 250 and 25 mg ai/L stock solutions. An aliquot (200 mL) of the 25 mg ai/L stock solution was dispersed with synthetic sewage (16 mL), allythiourea (ATU) (5 mL of a 1.16 g/L stock solution), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg ai/L. Similarly, aliquots (200 mL) of the 250 mg ai/L and 2500 mg ai/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg ai/L. The 1000 mg ai/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

The pH of the test item stock solutions were measured using a a WTW pH/Oxi 340I pH and dissolved oxygen meter (see Table 6 - attached background material)) and adjusted to between pH 7.0 to pH 8.0.

The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on 11 July 2012 and 16 July 2012 for the range-finding tests from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.


PREPARATION OF INOCULUM:
The activated sewage sludge sample used to measure total respiration was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 8.0 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained.

The suspended solids concentration was equal to 3.0 g/L prior to use.

SYNTHETIC SEWAGE:
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
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 1 litre of water with the aid of ultrasonication.

In the range-finding test for total respiration, the pH of the synthetic sewage stock was pH 7.1 and in the range-finding test for heterotropic respiration the pH of the synthetic sewage stock used to feed the activated sewage sludge was 7.1 and the pH of the synthetic sewage stock used in the test was 7.0. The pH values were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

Not stated.

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 20 ± 2ºC.

pH:

The pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Nominal and measured concentrations:

Nominal test concentrations in range-finding test to measure total respiration and range-finding test to measure heterotrophic respiration were:
10, 100 and 1000 mg (ai)/L.

Details on test conditions:

PREPARATION OF TEST SYSTEM:
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as described in 'details of test solutions' section. Finally two further control vessels were prepared.

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/l and 2.0 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.


TEST MEDIUM / WATER PARAMETERS
The test water used for the range-finding tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (at concentrations of 3.2, 10 and 32 mg/L).

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Total Respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Total respiration

Details on results:

RANGE-FINDING TESTS

TOTAL RESPIRATION:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4. Observations made on the test preparations throughout the study are given in Table 5. The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2.

See attached background material for all tables.

The coefficient of variation of oxygen uptake in the control vessels was 4.0% and the specific respiration rate of the controls was 24.01 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were at or above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.

No statistically significant toxic effects were shown at all of the test concentrations employed.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L.

HETEROTROPIC RESPIRATION:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 8. The pH values of the test preparations at the start and end of the exposure period are given in Table 9. Observations made on the test preparations throughout the study are given in Table 10. The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 7.

See attached background material for all tables.

The coefficient of variation of oxygen uptake in the control vessels was 2.7%. The validation criterion has therefore been satisfied.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.

The relatively large increase in respiration rate observed in the test vessels, see Table 8, was considered to be due to the possible hormetic response of the activated sewage sludge micro-organisms to the test material, i.e. the stimulation of biological activity due to the presence of test material at concentrations below its toxic level.

No statistically significantly toxic effects were shown at the test concentrations of 100 and 1000 mg ai/L, however statistically significantly effects were shown at the test concentration of 10 mg ai/L. However there was not a decrease in the oxygen consumption value for the 10 mg ai/L test concentration compared to the controls and the statistical difference was not as a result of inhibition but as a result of the increase in the respiration rate in the vessel compared to the controls. Therefore the No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg ai/L.

NITRIFICATION RESPIRATION:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 11 (see attached background material).

The coefficient of variation of oxygen uptake in the control vessels was 9.8%. The validation criteria have therefore been satisfied.

No statistically significant toxic effects were shown at all of the test concentrations employed.

Results with reference substance (positive control):

TOTAL RESPIRATION:
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 1 - see attached background material).

The following results were derived:
3 hour EC20: 3.1 mg/L
3 hour EC50: 8.8 mg/L (95% Confidence Limits: 7.0 - 11 mg/L)
3 hour EC80: 25 mg/L

The validation criterion for the reference item EC50 value was satisfied.

HETEROTROPIC RESPIRATION:
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 2 - see attached background material).

The following results were derived:
3 hour EC20: 6.1 mg/L
3 hour EC50: 13 mg/L (95% Confidence Limits: 11 - 15mg/L)
3 hour EC80: 29 mg/L

The validation criterion for the reference item EC50 value was satisfied.

NITRIFICATION RESPIRATION:
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 3 - see attached background material).

The following results were calculated:
3 hour EC20: 0.10 mg/L
3 hour EC50: 1.0 mg/L (95% Confidence Limits: 0.6 - 1.7 mg/L)
3 hour EC80: 11mg/L

The validation criterion for the reference item EC50 value was satisfied.

Discussion:

Based on the results of the two range-finding tests and given that no statistically significant toxic effects were shown at all of the test concentrations employed it was considered justifiable not to perform a definitive test.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the total respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg ai/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg ai/L.

Executive summary:

Introduction

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) ".

Methods

Activated sewage sludge was exposed to an aqueous solution of the test item at concentrations of 10, 100 and 1000 mg active ingredient (ai)/L (3 replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at a temperature of 20 ± 2°C with the addition of a synthetic sewage as a respiratory substrate and with and without the presence of specific nitrification inhibitor ATU.

The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the total respiration of activated sewage sludge gave a 3-Hour EC₅₀ value of greater than 1000 mg ai/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg ai/L.

The reference item gave a following 3 -Hour EC₅₀ values and 95% confidence limits.

	Total Respiration		Heterotropic Respiration		Nitrification Respiration
	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx(3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC20	3.1	-	6.1	-	0.10
EC50	8.8	7.0 – 11	13	11 – 15	1.0	0.6 – 1.7
EC80	25	-	29	-	11

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to Annex 3 of the CSR or IUCLID Section 13 for justification of read-across within the HEBMP category.

Reason / purpose for cross-reference:

read-across source

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Total respiration

Description of key information

No data are available for the registration substance. Data are available for the sodium salt form. IC₅₀ (3 h) >1000 mg/L act. ingr. (>800 mg/L as active acid equivalent, in which 57% w/w was present as cyclic form). No NOEC or IC₁₀ was derived by the study authors, but it should be noted that at 1000 mg/L act. ingr. loading rate, less than 10% inhibition was recorded in all replicates.

Additional information

The study was conducted in accordance with the appropriate guideline and with GLP.

The acid and sodium salts in the HEBMP category are freely soluble in water. The HEBMP anion can be considered fully dissociated from its sodium cation when in dilute solution. Under any given conditions, the degree of ionisation of the HEBMP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was HEBMP-H, HEBMP-xNa, or another salt of HEBMP.

 

Therefore, when a salt of HEBMP is introduced into test media or the environment, the following is present (separately):

1. HEBMP is present as HEBMP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the media and not whether HEBMP-xNa salt, HEBMP-H (acid form), or another salt was used for dosing.
2. Disassociated sodium cations. The amount of sodium present depends on which salt was added.
3. It should also be noted that divalent and trivalent cations would preferentially replace the potassium ions. These would include calcium (Ca²⁺), magnesium (Mg²⁺) and iron (Fe³⁺).