Reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide

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:

1997-07-02 to 1997-07-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

April 04, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

May 05, 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the limited water solubility of the test item the following dosage of the test substance was chosen:
Test substance amounts of 5, 16, 50, 160, and 500 mg were directly weighed by means of an analytical balance into 1000-mL glass flasks containing 284 mL of deionized water. The test substance was mixed with the deionized water by ultrasonic treatment for five minutes and intense stirring for four hours at room temperature in the dark to reach the maximum concentration of dissolved test substance (practically achievable maximum concentration up to the limit of water solubility) and in case of excess of test substance to reach the saturation equilibrium. The 4-hour stirring was chosen based on the results of a pre-test performed within RCC Project No. 657101 within which no significant difference in the test article concentration was obtained analytically in filtrates of solutions with 2 and 24-hour stirring times. Immediately prior to test start (incubation), 16 mL of synthetic sewage feed and 200 mL of sludge inoculum was added, resulting in a final volume of 500 mL. Thus, the following nominal test substance concentrations were tested: 10, 32, 100, 320, and 1000 mg/L. Concentrations in excess of nominal 1000 mg test substance/L were not tested.

- Controls: To obtain the test media of the reference substance a stock solution of 3,5-dichlorophenol was prepared according to the test guidelines: 0.5 g of 3,5-dichlorophenol was dissolved in 10 mL 1 mol/L NaOH and diluted to about 30 mL with bidistilled water. Excess of NaOH was neutralized with 0.5 mol/L H2SO4 to the point of incipient precipitation. Thereafter, the mixture was made up to one litre with bidistilled water. The pH was determined to be 7.8, and the final concentration amounted to 500 mg/L. Aliquots of this stock solution were mixed with deionized water in the respective 1000-mL glass flasks. Immediately prior to test start (incubation), the flasks were filled up to 500 mL final volume. The following nominal concentrations of the reference substance were tested: 3.2, 10, 32, and 100 mg/L.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic sewage. The sludge was washed by centrifugation, the supernatant liquid phase was decanted and the solid material resuspended in tap water. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet sludge to its dry weight determined. Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to a mixed liquor of suspended solids (MLSS) of 2 g per litre (on dry weight basis).
- Method of cultivation: During holding, the sludge was fed daily, starting four days prior to use, with 50 mL synthetic sewage per litre and was kept at room temperature under continuous aeration until use.
- Initial biomass concentration: Immediately before use, the dry weight of the activated sludge was determined to be 2 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

22 °C at the start and 21 °C at the end of incubation

pH:

The pH of the activated sludge inoculum was 7.6.

Dissolved oxygen:

The concentration of dissolved oxygen did not drop below 2.5 mg/L during incubation, and was at least 6.5 mg/L prior to measurement of the respiration rate.

Salinity:

no data, however the used synthetic sewage contained reliable amounts of NaCl, CaCl2, MgSO4 and K2HPO4

Nominal and measured concentrations:

nominal: 5, 16, 50, 160, and 500 mg/L

Details on test conditions:

EXPERIMENTAL CONDITIONS
Immediately prior to test start (incubation), the activated sludge (inoculum) with a sludge concentration of 2.0 g/L dry weight (MLSS) was first added to an inoculum control, then in time intervals of about ten minutes (an arbitrary but convenient interval) to the test solutions of the reference substance 3,5-dichlorophenol, to an additional internal inoculum control, to the suspensions of the test substance, and at least to the final inoculum control.

During the incubation period of exactly three hours all test media were continuously aerated with compressed air at an air-flow of about one litre per minute.

MEASUREMENT of the RESPIRATION RATE
For measurement of the respiration rate a well mixed sample of each test flask was poured into BOD-flask after exactly three hours incubation time, and was further aerated. The oxygen concentration was measured with an oxygen electrode, and was continuously recorded for about ten minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the most linear part of the respiration curve, or in case of low oxygen consumption over a period of about ten minutes.

DETERMINATION of the INHIBITION RATE
The inhibitory effect of the test substance or of the reference substance on the respiration rate (oxygen consumption per minute) is expressed as a percentage of the mean respiration rate of the two controls measured at the start and at the end of the test series.

As far as possible, the 3-hour EC50 and EC20 and their 95 %-confidence limits were calculated by Probit analysis. All biological results and the calculations of the EC-values were based on nominal concentrations.

MEASUREMENT of pH, DISSOLVED OXYGEN and WATER TEMPERATURE
The pH was determined in the freshly prepared test medium of controls and all test concentrations of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.). At the end of the incubation period the pH-values were determined in all test media. The oxygen concentration war checked at the start and at the end of the 3-hour incubation period in all test media. The water temperature was measured in one control medium at the start and at the end of the incubation period.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: clearly higher than the limit of water solubility

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: clearly higher than the limit of water solubility

Details on results:

TOXICITY of the TEST ITEM:
In comparison to the inoculum controls the respiration rate of the activated sludge was not significantly inhibited (< 15 %) at any of the nominal concentrations of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) in the range from 10 to 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. From the appearance of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) in the final test medium, an obvious excess of test article on the surface of the test media was present at 100, 320, and 1000 mg/L. At the two lower concentrations of 32 and 10 mg/L, white suspensions were noted. Thus, the saturation concentration (equilibrium) under the present test conditions was reached and resulted in less than 20 and 50 % inhibition after three hours contact time. The 3-hour EC20 and EC50 are clearly higher than the limit of water solubility under the present test conditions, or > 1000 mg/L nominal.

VALIDITY of the TEST
The test is regarded as valid since the variance between the two inoculum control samples (start, end) was less than 15 % (2.5 %), and the 3-hour EC50 of the positive control 3,5-dichlorophenol was 23.7 mg/L. Both values are within the guideline-recommended values.

Results with reference substance (positive control):

In comparison to the inoculum controls the respiration rate of the activated sludge was not significantly inhibited (3.6 and 9.3 %) at the two lower nominal concentrations of 3.2 and 10 mg/L. At the next higher nominal concentrations of 32 and 100 mg/L, the respiration rate was inhibited by 76.2 and 88.2 %, respectively. From these inhibition rates, the 3-hour EC50 for the positive control 3,5-dichlorophenol was calculated by the Probit analysis to be 23.7 mg/L (the 95 % confidence limits were not calculated).

Executive summary:

Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) was assessed for its toxicity to aerobic wastewater microorganisms of activated sludge in a 3-h respiration inhibition test according to OECD guideline 209, April 04, 1984 and EU method C11, May 05, 1988. The substance was tested up to and including a nominal concentration of 1000 mg/L which is well above the water solubility limit.

In comparison to the inoculum controls, the respiration rate of the activated sludge was not significantly inhibited (< 15 %) at any of the nominal substance concentrations in the range from 10 to 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. From the appearance of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) in the final test medium, an obvious excess of test article on the surface of the test media was present at 100, 320, and 1000 mg/L. At the two lower concentrations of 32 and 10 mg/L, white suspensions were noted. Thus, the saturation concentration (equilibrium) under the present test conditions was reached.

The 3-hour EC₂₀ and EC₅₀ are clearly higher than the limit of water solubility under the present test conditions, or > 1000 mg/L nominal.

From the results of this test it can be stated that the substance poses no hazard to the microbiological activity in sewage treatment plants.

The 3-h EC₅₀ of the positive control substance 3,5-dichlorophenol was within the accepted range, thus, confirming suitability of the activated sludge used. This toxicity study is classified as acceptable and satisfies the requirements of the OECD guideline 209 and the EU method C11.

Description of key information

A reliable, relevant and adequate study according to OECD guideline 209, April 04, 1984 and EU method C11, May 05, 1988 is available.
The 3-h EC50 for activated sludge based on respiration inhibition was > 1000 mg/L nominal. It can be stated that substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" poses no hazard to the microbiological activity in sewage treatment plants. Therefore, no PNEC STP will be derived.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) was assessed for its toxicity to aerobic wastewater microorganisms of activated sludge in a 3-h respiration inhibition test according to OECD guideline 209, April 04, 1984 and EU method C11, May 05, 1988. The substance was tested up to and including a nominal concentration of 1000 mg/L which is well above the water solubility limit.

In comparison to the inoculum controls, the respiration rate of the activated sludge was not significantly inhibited (< 15 %) at any of the nominal substance concentrations in the range from 10 to 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested. From the appearance of substance "reaction mass of calcium hydrogen phosphonate and dialuminium tricalcium hexaoxide" (90 - 95 % a.i.) in the final test medium, an obvious excess of test article on the surface of the test media was present at 100, 320, and 1000 mg/L. At the two lower concentrations of 32 and 10 mg/L, white suspensions were noted. Thus, the saturation concentration (equilibrium) under the present test conditions was reached and resulted in less than 20 and 50 % inhibition after three hours contact time.

The 3-h EC₂₀ and EC₅₀ are clearly higher than the limit of water solubility under the present test conditions, or > 1000 mg/L nominal.

The 3-h EC₅₀ of the positive control substance 3,5-dichlorophenol was within the accepted range, thus, confirming suitability of the activated sludge used. This toxicity study is classified as acceptable and satisfies the requirements of the OECD guideline 209 and the EU method C11.