p-phenylene diisocyanate

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:

06 Jun 2018 to 21 Jun 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))

Version / remarks:

OECD Guideline 209. "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)”, adopted 22 July 2010.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

No further details specifed in the study report.

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

The batch of PPDI tested consisted of white chips and had a purity of 99.8%. 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. Therefore, weighed amounts were added to the amber 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. Optimal contact between test item and test organisms was ensured by applying continuous aeration and stirring.
Any residual volumes were discarded.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas','s -Hertogenbosch, Heeswijk-Dinther, 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. 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 7.0 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
Rationale: Recognized by international guidelines as the recommended test system.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified

Test temperature:

18 and 21°C

pH:

6.8 and 7.8.

Dissolved oxygen:

Not applicable - respiration inhibition study

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Final Test – Test Concentrations
Paraphenylene diisocyanate (PPDI) Five concentrations: 100, 180, 320, 560 and 1000 mg/L.

Details on test conditions:

Testing Strategy and Experimental Design
Combined Limit/Range-Finding Test
In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested.
The highest concentration was tested in triplicate, lower concentrations consisted of one replicate. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested.
Test procedure and conditions were similar to those applied in the final test with the following exceptions:
-The pH of the sludge was 5.8 on the day of testing. The sludge was buffered to a pH of 7.5 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the respiration rate of the sludge was determined to verify the quality of the sludge. The mean respiration rate of the sludge was 27 mg O2/g.h and within the validity criterion.

Final Test
Replicates: 5 replicates per test group and 6 replicates for the control.
Control: Test medium without test item, treated in the same way as the test item solutions.

Test Procedure and 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.
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.
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.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

Details on results:

RESULTS
Combined Limit/Range-Finding Test
The combined limit/range-finding showed no inhibition of the respiration rate at 10 and 100 mg/L. At 1000 mg/L, 21% inhibition of the respiration rate was observed. Therefore, the expected EC50 was above 1000 mg/L. There was no oxygen uptake from abiotic processes.
Since the effect observed at 1000 mg/L was statistically significant a final test was performed to determine the NOEC.
All test conditions 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 or below 320 mg PPDI per liter. Replicate 5 at 560 mg/L was determined to be an outlier (see Appendix 3) and was not used in the statistical calculations.

Results with reference substance (positive control):

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

Reported statistics and error estimates:

Test Item - Shapiro-Wilk´s Test on Normal Distribution
Number of residuals = 30
Shapiro-Wilk´s W = 0.961
p(W) = 0.332
p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution. Normality check was passed (p > 0.01).

Test Item - Levene´s Test on Variance Homogeneity (with Residuals)
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled. A parametric multiple test is advisable.

Test Item - Determination of NOEC: Williams Multiple Sequential t-test Procedure.
A NOEC of 320 mg/L is suggested by the program.

Final Test – Overview of the 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	33.63	22.42
T1	100	35.08	23.38	-4.30
T2	180	31.14	20.76	7.40
T3	320	32.26	21.51	4.07
T4	560	29.24	19.49	13.06*
T5	1000	25.52	17.02	24.10*

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

* Statistically significantly different compared to control.

Effect Parameters

Parameter	PPDI Concentration (mg/L)
NOEC	320
EC10	401 (264 - 609)
EC20	823 (601 - 1126)
EC50	>1000

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

Combined Limit/Range-Finding Test – Respiration Rate/Inhibition, pH Values

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C 1	0	7.7	7.8	37.49	24.99
C 2	0	7.7	7.8	25.91	17.27
C 3	0	7.6	7.7	34.72	23.15
C 4	0	7.6	7.7	34.91	23.27
C 5	0	7.6	7.6	36.55	24.37
C 6	0	7.6	7.6	32.76	21.84
C Mean				33.72	22.48 (RC)
SD				4.16	2.77
CV (%)				12	12
R 1	1.0	7.7	8.0	29.65	19.77	12.08
R 2	3.2	7.7	8.2	16.92	11.28	49.83
R 3	10	7.7	8.2	10.18	6.79	69.81
R 4	32	7.7	8.2	4.53	3.02	86.57
T 1	10	7.7	7.8	34.76	23.17	-3.07
T 2	100	7.7	7.8	35.89	23.93	-6.42
T 3a	1000	7.6	7.8	26.42	17.61	21.66
T 3b	1000	7.6	7.7	27.12	18.08	19.58
T 3c	1000	7.6	7.8	26.04	17.36	22.78
T3 Mean				26.53	17.68 (RT)*	21.34 (IT)
TA	1000	7.6	7.4	0.00#	0.00	100.00

C:          Control

R:          Reference item, 3,5-dichlorophenol

T:          Test item, PPDI   

TA:        Abiotic control of PPDI       

SD:       Standard deviation             

CV:       Coefficient of variation                       

RT:         Total respiration with PPDI

RC:        Total respiration in the control

IT:           % inhibition of total respiration relative to RC

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

*                            Statistically significantly different compared to control 

#             No respiration, therefore expressed as 0 mg O2/L.h

Final Test –Respiration Rate/Inhibition, pH Values

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C 1	0	7.3	7.2	31.00	20.67
C 2	0	7.3	7.1	34.88	23.25
C 3	0	7.3	7.0	31.62	21.08
C 4	0	7.3	7.0	34.91	23.27
C 5	0	7.3	6.9	35.27	23.51
C 6	0	7.3	6.9	34.10	22.73
C Mean				33.63	22.42 (RC)
SD				1.85	1.23
CV (%)				5	5
R 1	1.0	7.3	7.6	27.06	18.04	19.54
R 2	3.2	7.3	7.8	15.20	10.13	54.80
R 3	10	7.3	7.7	8.79	5.86	73.86
R 4	32	7.3	7.7	3.38	2.25	89.95
T 1a	100	7.3	7.0	34.58	23.05	-2.82
T 1b	100	7.3	7.1	33.02	22.01	1.81
T 1c	100	7.3	7.1	34.05	22.70	-1.25
T 1d	100	7.3	-	36.98	24.65	-9.96
T 1e	100	7.3	-	36.75	24.50	-9.28
T 1 Mean				35.08	23.38 (RT)	-4.30 (IT)
T 2a	180	7.3	7.1	32.34	21.56	3.84
T 2b	180	7.3	7.1	28.80	19.20	14.36
T 2c	180	7.3	7.1	29.36	19.57	12.70
T 2d	180	7.3	7.1	31.92	21.28	5.08
T 2e	180	7.3	7.0	33.29	22.19	1.01
T 2 Mean				31.14	20.76 (RT)	7.40 (IT)

C:           Control

SD:       Standard deviation

CV:        Coefficient of variation

R:           Reference item, 3,5-dichlorophenol

T:            Test item, PPDI

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

RC:        Total respiration in the control

RT:         Total respiration with PPDI

IT:           % inhibition of total respiration relative to RC

-:             No pH measurement, see deviation inError! Reference source not found.

           
Final Test – Respiration Rate/Inhibition, pH Values (continued)

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
T 3a	320	7.3	6.9	31.01	20.67	7.79
T 3b	320	7.3	6.9	34.72	23.15	-3.24
T 3c	320	7.3	7.0	27.79	18.53	17.37
T 3d	320	7.3	6.9	35.91	23.94	-6.78
T 3e	320	7.3	6.9	31.87	21.25	5.23
T 3 Mean				32.26	21.51 (RT)	4.07 (IT)
T 4a	560	7.3	7.0	23.97	15.98	28.72
T 4b	560	7.3	6.8	33.31	22.21	0.95
T 4c	560	7.3	7.0	29.22	19.48	13.11
T 4d	560	7.3	6.9	30.45	20.30	9.46
T 4e	560	7.3	7.0	5.94 2	3.96 2	82.34
T 4 Mean				29.24	19.49 (RT)*	13.06 (IT)
T 5a	1000	7.3	6.9	23.72	15.81	29.47
T 5b	1000	7.3	6.9	29.05	19.37	13.62
T 5c	1000	7.3	6.9	29.19	19.46	13.20
T 5d	1000	7.3	6.9	25.72	17.15	23.52
T 5e	1000	7.3	6.9	19.94	13.29	40.71
T 5 Mean				25.52	17.02 (RT)*	24.10 (IT)

T:            Test item PPDI

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

RT:         Total respiration with PPDI

IT:           % inhibition of total respiration relative to RC

*:            Statistically significantly different compared to control

2:            This replicate is an outlier and is not used for calculations of the NOEC and ECx values. 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test Paraphenylene diisocyanate (PPDI) was not toxic to waste water bacteria (activated sludge) at or below a concentration of 320 mg/L (NOEC).
The EC10 was 401 mg/L (95% confidence interval: 264 - 609 mg/L).
The EC20 was 823 mg/L (95% confidence interval: 601 - 1126 mg/L).
The EC50 was above 1000 mg/L.

Executive summary:

The objective of the study was to evaluate Paraphenylene diisocyanate (PPDI) for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC).

 

The influence of PPDI 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 on the OECD guideline No. 209, 2010.

 

The batch of PPDI tested consisted of white chips and had a purity of 99.8%. 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. 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 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 100 mg/L to 1000 mg/L and increasing with a factor 1.8. 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 320 mg PPDI per liter. At the two higher concentrations the inhibitory effect of PPDI on aerobic waste water (activated sludge) bacteria increased with increasing concentration, with 13% inhibition at 560 mg/L and 24% at 1000 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.

 

PPDI was not toxic to waste water (activated sludge) bacteria at or below a concentration of 320 mg/L (NOEC).

 

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

Parameter	PPDI Concentration (mg/L)
NOEC	320
EC10	401 (264 - 609)
EC20	823 (601 - 1126 )
EC50	>1000

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

Description of key information

Paraphenylene diisocyanate (PPDI) was not toxic to waste water bacteria (activated sludge) at or below a concentration of 320 mg/L (NOEC).

The EC10 was 401 mg/L (95% confidence interval: 264 - 609 mg/L).

The EC20 was 823 mg/L (95% confidence interval: 601 - 1126 mg/L).

The EC50 was above 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

320 mg/L

Additional information

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 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 100 mg/L to 1000 mg/L and increasing with a factor 1.8. 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 320 mg PPDI per liter. At the two higher concentrations the inhibitory effect of PPDI on aerobic waste water (activated sludge) bacteria increased with increasing concentration, with 13% inhibition at 560 mg/L and 24% at 1000 mg/L.

 

PPDI was not toxic to waste water (activated sludge) bacteria at or below a concentration of 320 mg/L (NOEC).

 

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

Parameter	PPDI Concentration (mg/L)
NOEC	320
EC10	401 (264 - 609)
EC20	823 (601 - 1126 )
EC50	>1000

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