Reaction mass of 1,2-dichloropropane and 3-chloropropene and 3,3-dichloropropene

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Relevant study conducted according to relevant OECD guideline and performed under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Deviations:

yes

Remarks:

The quality of the study was not affected by these deviations.

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

A 100g sample of PDC was obtained from the Aldrich Chemical Company (Milwaukee, Wisconsin). The sample was identified by lot # 06514HW with a reported purity of 99.6% by gas chromatography (GC) and structural confirmation by infrared analysis. The testing laboratory confirmed a purity of >99% by GC with flame ionization detection (FID) as well as the structural identity of PDC as 1,2-Dichloropropane by GC-mass spectrometry. The scanning range for the PDC test material was set from 50 to 200 atomic mass units (amu).

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

The microbial inoculum consisted of activated sludge mixed liquor collected from the Midland Municipal Wastewater Treatment Plant (Midland, Michigan) on 19 August 2002. This facility treats approximately three million gallons of wastewater per day, of which >90% is from domestic sources. Upon return to the laboratory, the activated sludge was washed twice with tap water, and dispersed in mineral medium. The mixed liquor suspended solids (MLSS) concentration of the activated sludge was determined to be 2240 +/- 40 mg/L (n=2) using a standard procedure. The activated sludge was aerated overnight.
The following day, the MLSS was determined to be 2080 +/- 80mg/l (n=4). The pH of the activated sludge was adjusted from 7.0 to 7.4 with 1N
NaOH.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

150 mg/L

Based on:

other: nominal concentration

Initial test substance concentration 2

Initial conc.:

50 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation 1

Parameter followed for biodegradation estimation:

DOC removal

Parameter followed for biodegradation estimation 2

Parameter followed for biodegradation estimation:

test mat. analysis

Remarks:

GC

Details on study design:

Reaction mixtures containing PDc were prepared in 1-liter glass bottles constructed with glass baffles. The vessels were sealed with caps equipped with two sampling ports. One port contained a sampling valve with a Luer lock fitting connected to Teflon tubing that permitted sampling of the reaction mixture. A second port was fitted with a plastic sleeve to insert a fiber optic probe for headspace oxygen measurements. The probe was protected in a #18 gauge stainless steel needle that was inserted through the sampling valve into the vessel headspace.
Preliminary studies indicated that even minimal aeration of the reaction mixtures resulted in extensive losses of PDC by volatilization. Further work showed that aerobic conditions should be maintained in a sealed test vessel by continuous mixing of the reaction mixtures and periodic additionof oxygen concentration in the headspace gas was depleted by biological activity.
Portions of activated sludge (240ml) were combined with 255 ml of mineral medium and adjusted to a final volume of 500 ml with aniline stock solution or water, as appropriate.
Test mixtures were prepared by adding 65µl of PDC (density 1.16 mg/µl) to duplicate vessels (designated A and B) to obtain a nominal concentration of 150 mg/l PDC. This concentration is equivalent to approximately 50 mg/l DOC (PDC contains 32% carbon). A previous study reported that the EC50 concentration for PDC in an OECD 209 Activated Sludge Respiration Inhibition Test was 520 mg/l. Thus, the initial PDC concentration in the present study was 3.5-fold lower than the reported EC50 concentration in activated sludge.
Duplicate abiotic controls (designated C and D) were prepared in a similar manner to follow abiotic degradation and/or volatilization of the test compound. The abiotic controls were prepared with 250 mg/l mercuric chloride added to the activated sludge to inhibit biological activity.
Biodegradation of 72 mg/l aniline (reference compound) was determined in positive control mixtures to confirm the viability of the inoculum (designated E and F).
Toxicity control mixtures (designated G and H) amended with 72 mg/l aniline and 150 mg/l PDC were used to determine whether PDC was inhibitory to the microbial inoculum.
Inoculum control mixtures (designated I and J) containing only the inoculated medium were included to allow measurement of background DOC concentrations in the reaction mixtures and possible interferences in the compound specific analysis by GC.
The oxygen concentration in the headspace and the pH of selected reaction mixtures were measured to ensure that aerobic conditions and the proper pH range were maintained. The reaction mixtures were continually mixed with a magnetic stirrer at 150 rpm and incubated at ambient temperature (22+/-1°C) for 28 days.
Reaction mixtures were sampled after 0 (3 hours after addition of PDC), 1, 2, 7, 14, 21, and 28 days to follow the degradation of PDC and aniline. Additional samples were collected following the conclusion of the experiment (day 31) for additional DOC analyses. A disposable 20ml plastic syringe was connected to the sampling port of the reaction vessel with a Luer lock fitting and approximately 25ml of the reaction mixture was removed. The sample was passed through a 0.45µm nylon membrane filter containing a glass fiber pre-filter. Filters were pre-rinsed with water prior to use. For samples collected on days 28 and 31, centrifugation was used to separate the sludge solids from the liquid phase prior to filtering in order to reduce excessive backpressures encountered in the filtering step. The filtrate was collected in a 40ml vial chilled in ice to minimize volatilization losses of PDC. Following collection of the filtrate, a 1ml subsample was removed and mixed with 1ml of 25% phosphoric acid solution in a 20ml headspace vial that was chilled in ice.
The pH of the reaction mixtures was monitored indirectly by measuring the pH of filtered samples following DOC analyses. This approach avoided volatilization losses for PDC that would have occured if the test vessels were opened to insert a pH probe. On day 10, 2.5ml of the pH 7.4 buffer solution used to prepared to mineral medium (solution I) was added to each of the reaction mixtures in order to maintain the pH of the reaction mixtures within the range of pH 6.5 to 8.0 as recommended in the test guidelines.
The oxygen concentration in the test vessel headspace gases was measured using a fiber optic probe. The probe was inserted through a sampling port in the vessel cap. Oxygen was routinely added to ensure that headspace concentrations of oxygen did not fall below 10%. The oxygen concentrations in the headspace gases were routinely replenished by the addition of 40 ml oxygen gas through the sampling tube directly into the reaction mixtures. The abiotic controls were typically amended with 40ml laboratory air since little oxygen consumption occurred in these mixtures. Following the addition of oxygen or air, the headspace gases were vented through a valve located in the vessel cap to avoid a pressure buildup in the vessels. All reaction mixtures were replenished with the same volume of gas to minimize variation in the losses of PDC by volatilization.

Reference substance

Reference substance:

aniline

Remarks:

Aldrich Chemical Company, lot #01408TI

Results and discussion

Preliminary study:

Preliminary studies indicated that even minimal aeration of the reaction mixtures resulted in extensive losses of PDC by volatilization. Further work showed that aerobic conditions should be maintained in a sealed test vessel by continuous mixing of the reaction mixtures and periodic additionof oxygen concentration in the headspace gas was depleted by biological activity.

% Degradation

Parameter:

% degradation (test mat. analysis)

Value:

7

Sampling time:

28 d

Details on results:

PDC did not show evidence of inherent biodegradability using a modification of the Zahn-Wellens/EMPA test. The reaction mixtures were continually mixed at 22 +/- 1°C and incubated in closed vessels to minimize the loss of PDC due to volatilization. Oxygen concentrations in the headspace of the vessels was monitored and oxygen was added as necessary to ensure that aerobic conditions were maintained. No measurable biodegradation of PDC was observed during the 28-day test. PDC measurements in the reaction mixtures showed no loss of the test compound in the viable mixtures (test and toxicity control mixtures) compared to the aerobic controls. Note that abiotic control C showed a 35% loss of PDC after 28 days compared to a 6% loss of PDC in abiotic control D. These results suggest leaks of headspace gases in vessel C may have contributed to the 35% loss of PDC. Test mixtures A and B showed a 7% loss of PDC and the toxicity controls G and H showed an 8% loss of PDC after 28 days. Overall the results of the PDC analyses indicate little difference in PDC concentrations between viable and abiotic control mixtures over time. Thus, PDC did not biodegrade under the conditions of this test.
DOC measurements confirmed that PDC did not biodegrade in the test. Little difference was noted in DOC measurements between the viable and abiotic control mixtures in the experiment. Note that DOC results were not available for the day 21 and day 28 sampling points. However, DOC measurements of additional samples collected three days after the conclusion of the test (day 31) showed DOC concentrations of 34.4 +/- 1.8 mg/l in the viable mixtures and 38.2 +/-6.9 mg/l in the abiotic controls. These results confirmed that the PDC did not biodegrade in the test mixtures.
Decrease in pH were noted in each of the reaction mixtures over time. At the conclusion of the experiment, the pH of viable reaction mixtures containing PDC were approximately pH 6.4. The test guidelines recommend that the pH of the reaction mixtures be maintained in the range of pH 6.5 to 8.0. Thus, the pH of the reaction mixtures was maintained within an acceptable range during the study.
The headspace oxygen concentrations for the Test Mixtures (A and B) did not fall below 12% during the experiment. Assuming that oxygen concentrations in the liquid phase were in equilibrium with the headspace concentrations in the constantly stirred mixtures, the 12% oxygen concentration in the headspace correspond to approximately 5mg/l dissolved oxygen in the liquid phase. Activated sludge treatment processes are typically supplied with sufficient oxygen to maintain dissolved oxygen concentrations of at least 2mg/l. Thus, sufficient oxygen was available to maintain aerobic conditions in the reaction mixtures.

BOD5 / COD results

Results with reference substance:

Biodegradation of aniline in the positive control mixtures reached 87% after seven days and 96% after 14 days. The test is considered valid if biodegradation of aniline reaches 70% in 14 days. The extensive degradation of aniline in the positive controls confirmed the viability of the micobial inoculum. In addition, extensive degradation of aniline in the toxicity controls (98% after 14 days) indicated that PDC was inhibitory to the microbial inoculum under the conditions of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

1,2-Dichloropropane did not meet the criteria of inherent biodegradability under the conditions of a modified OECD Method 302B test.

Executive summary:

The inherent biodegradability of 1,2 -dichloropropane (propylene dichloride or PDC) was assessed using a modification of the Zahn-Wellens/EMPA test (OECD Method 302B). Reaction mixtures were prepared by dispersing activated sludge from a municipal wastewater treatment plant in a standard mineral medium. Reaction mixtures were amended with 150mg/L PDC and incubated in closed vessels to minimize the loss of PDC due to volatilization. oxygen concentrations in the headspace of the vessels were monitored and oxygen gas was added as necessary to ensure that aerobic conditions were maintained. The reaction mixtures were continuously mixed and incubated at 22 +/- 1°C for 28 days.

Compound specific analyses of PDC in the liquid phase of the reaction mixtures by gas chromatography with flame ionization (GC-FID) showed little difference in PDC concentrations over time between viable and abiotic control mixtures (biologically inhibited with mercuric chloride). Thus, PDC did not biodegrade under the conditions of this test. Measurement of dissolved organic carbon (DOC) concentrations in the reaction mixtures confirmed the results of the GC-FID analyses.

Aniline (reference compound) was extensively degraded in positive control mixtures (96% in 14 days), thereby confirmed the viability of the microbial inoculum. Similar extensive degradation of aniline in the presence of PDC (98%) in 14 days) demonstrated that PDC was not inhibitory to the inoculum under the test conditions.