Dialkyl C18 and C18-unsaturated phosphonates

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From2017-06-01 to 2017-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dialkyl C18 and C18-unsaturated phosphonates

EC Number:

701-298-1

Cas Number:

64051-29-2

Molecular formula:

Not applicable for a UVCB Substance

IUPAC Name:

Dialkyl C18 and C18-unsaturated phosphonates

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

no

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

The batch of Alkenyl phosphonate tested was a colourless liquid UVCB (Unknown, of Variable Composition, or of Biological origin). No correction was made for the purity/composition of the test item.
Since the test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L, weighed amounts were added to the dark brown 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 and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.

- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.6 on the day of testing. 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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

No data

Test temperature:

20 °C to 21 °C

pH:

7.2 to 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) and to maintain the sludge flocs in suspension

Salinity:

not applicable, fresh water

Nominal and measured concentrations:

Nominal concnetration: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- 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.
- No. of vessels per concentration (replicates):
Control: 6
Reference: 4
Low and medium dose: 1
Hih dose: 3
Abiotic control: 1

- Biomass loading rate: 3 g/L

TEST MEDIUM
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

Synthetic medium (=sewage feed)
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 Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

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.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid?: Yes

Any other information on results incl. tables

Results: Respiration Rate/Inhibition, pH Values

Replicate	Loading rate (T) Concentration (R) (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C 1	0	7.6	7.9	38.85	25.90
C 2	0	7.6	7.8	28.91	19.27
C 3	0	7.5	7.6	42.43	28.29
C 4	0	7.5	7.7	35.35	23.57
C 5	0	7.5	7.6	39.16	26.11
C 6	0	7.5	7.7	35.22	23.48
C Mean				36.65	24.44 (RC)
SD				4.65	3.10
CV (%)				13	13
R 1	1.0	7.6	8.0	26.89	17.93	26.64
R 2	3.2	7.6	8.2	17.68	11.79	51.76
R 3	10	7.6	8.1	9.35	6.23	74.49
R 4	32	7.6	8.1	6.31	4.21	82.78
T 1	10	7.5	7.6	37.49	24.99	-2.28
T 2	100	7.5	7.6	35.35	23.57	3.56
T 3a	1000	7.4	7.5	38.33	25.55	-4.57
T 3b	1000	7.4	7.5	36.51	24.34	0.39
T 3c	1000	7.4	7.7	30.38	20.25	17.12
T3 Mean				35.07	23.38 (RT)	4.31 (IT)
TA	1000	7.4	7.2	0.00^#	0.00^#	100.00

C:          Control                                                                            ¹             

R:          Reference item, 3,5-dichlorophenol   

T:          Test item, Alkenyl phosphonate                                       

TA:       Abiotic control of Alkenyl phosphonate                                         

SD:       Standard deviation                                                           

CV:       Coefficient of variation                                                   

#:           No respiration, therefore expressed as 0 mg O₂/L.h (see paragraph 6.1)

¹             The amount of suspended solids in the final test mixture was 1.5 g/L.

R_C:         Total respiration in the control

R_T:         Total respiration with Alkenyl phosphonate

I_T:          % inhibition of total respiration relative to R_C

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test Alkenyl phosphonate was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).
The ELR50 was above 1000 mg/L.

Executive summary:

The objective of the study was to evaluate Alkenyl phosphonate for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the ELR₅₀and/or the no-observed effect loading rate (NOELR).

The influence of Alkenyl phosphonate 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 Alkenyl phosphonate tested was a colourless liquid (UVCB). No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L, volumes corresponding to final concentrations of 10, 100 and 1000 mg/L have been added to the test-bottles containing Milli-RO water. The test item – Milli-RO water mixtures were magnetically stirred for a short period of 30 minutes and subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. 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.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested.Responses were compared to the controls.

Nostatistically significant inhibition of the respiration rate of the sludge was recordedat a loading rate of 1000 mg Alkenyl phosphonate per litre.Therefore, the ELR₅₀was above the highest concentration tested (1000 mg/L). There was no oxygen uptake from abiotic processes.

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.

Under the conditions of this present test, Alkenyl phosphonate was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR).

The ELR₅₀was above 1000 mg/L.