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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under guideline conditions and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 835.3110 (Ready Biodegradability)
Version / remarks:
1998
Qualifier:
according to guideline
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Version / remarks:
1992
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test Substance: Piperazine
Chemical Name: 1,4-diazacyclohexane
Test Substance Supplier: Sigma-Aldrich
Lot/Batch #: 1542OMC
Description: Solid, white to yellow crystals
Purity: 99.9% (by gas chromatography)
Compound Stability: Not determined
CAS #: 110-85-0
Molecular Formula: C4H10N2
MolecularWeight: 86.14
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
The microbial inocolum consisted of activated sludge mixed liquor, from a wastewater treatment plant with predominantly (95%) is from domestic sources. The (non-adapted) mixed liquor was collected the day before the test, and continuously aerated until used.
The Mixed liquor was mixed with mineral medium, to yield a final mixed liquor suspended solids concentration of 30 mg/L in the test. The initial pH was 7.57, and was adjusted to 7.40 using HCl.
Duration of test (contact time):
28 d
Initial conc.:
27.9 mg/L
Based on:
act. ingr.
Initial conc.:
51.8 mg/L
Based on:
ThOD/L
Parameter followed for biodegradation estimation:
CO2 evolution
Parameter followed for biodegradation estimation:
O2 consumption
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Measurements of headspace O2 and CO2 were recorded at six-hour intervals over the test period. At day 28 the aqeous phase was sampled for final DOC, nitrate and nitrite concentrations. The final pH was also recorded at day 28.
Reference substance:
aniline
Key result
Parameter:
% degradation (O2 consumption)
Value:
65
St. dev.:
27.4
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
39.3
St. dev.:
26.5
Sampling time:
28 d
Key result
Parameter:
% degradation (CO2 evolution)
Value:
70.2
St. dev.:
34.9
Sampling time:
28 d
Details on results:
See ilustration for result tables
Biodegradation of Piperazine
The results for biodegradation of piperazine and the reference substance (aniline) are summarized in Tables 4-6. Biodegradation of piperazine required 18.7 and 20.9 days for onset (DO2 > 10% of ThOD) in the replicate TestMixtures , and the course of degradation followed two distinct trends thereafter. The percentage of piperazine biodegradation based on BOD, CO2 evolution, and DOC removal averaged 65.3 ± 27.4%, 39.3 ± 26.5%, and 70.2 ± 34.9% (mean ± 1SD), respectively, at the end of the 28-day test. The average time for onset of piperazine biodegradation
was 19.9 days, and the 60% DO2 pass criterion was exceeded within 6 days thereafter (Figure 2). Thus, piperazine biodegradation exceeded the OECD-specified pass level of 60% DO2 within a 10-day window, and also exceeded the 70% DDOC pass level for DOC-based tests (i.e., OECD 301A) after 28 days. The measurement of extensive CO2 evolution further confirmed that piperazine was mineralized in this test, and not merely converted to intermediate degradation products (Table 5). Therefore, piperazine can be regarded as meeting the OECD pass criteria for ready biodegradability in the Manometric Respirometry test.
Although the study design did not include Toxicity Control reaction mixtures, which measure biodegradation of the reference substance in combination with the test substance, piperazine appeared to be slightly inhibitory to the microbial inoculum used in this test. Prior to onset of biodegradation, gross respiration of both oxygen and CO2 was somewhat suppressed in the piperazine TestMixtures relative to the Inoculum Blanks. This is evidenced by the initial series of negative DO2 and DCO2 values observed in the degradation curves (Figures 2 and 3). This inhibition of the inoculum may have contributed to the extended lag period, and imprecision in percentage biodegradation observed among the replicate piperazine TestMixtures.
The biodegradation of piperazine in replicate TestMixtures differed by >20%, as evidenced from BOD, CO2 evolution, and DOC removal at the end of the 28-day test. This difference among replicates is believed to be attributed to differences in the piperazine-degrading populations in these Test Mixtures , and not to imprecise or invalid test conditions. Over the course of the test, the piperazine Test Mixture replicate A was noted to be visibly less turbid than replicate B, indicating that the piperazine-degrading population in replicate B had grown to a much higher density at a more rapid rate than in replicate A. This difference in the growth rate of these piperazine-degrading populations is evidenced in the oxygen consumption and CO2 evolution curves for these replicates (Figures 2 and 3). The degradation curves for replicate A indicate an earlier onset of biodegradation, but a slower and notably linear growth of the degrading population. Conversely, the degradation curves for replicate B indicate a rapid and logarithmic growth of the piperazine -degrading microorganisms. The pH of these reaction mixtures also differed notably after 28 days, with replicate B being more acidic (pH = 6.93) than replicate A (pH = 7.21) and any of the Inoculum Blank and Positive Controlmixtures (pH range of 7.32–7.76).

Results with reference substance:
BOD:93.9 ± 1.0%, CO2 evolution 64.0 ± 1.5%, and DOC removal 90.3 ± 1.9%

Table 5. Summary of Biodegradation Based on Carbon Dioxide Evolution (D-CO2)

Reaction

Mixtures

Time to achieve (Days)

D-CO2 (%) at

10-d Window

Maximum D-CO2 (%)

Over 28 days

10%D-CO2

60% D-CO2

Aniline

Replicate A

Replicate B

Mean

 

 

 

 

3.8

19.6

50.7

65.1

4.3

22.2

50.5

62.9

4.1

20.6

50.6 ± 0.8

64.0 ± 1.5

Piperazine

Replicate A

Replicate B

Mean

 

 

 

 

23.2

N/A

N/A

20.6

21.9

N/A

N/A

58.0

21.9

N/A

N/A

39.3 ± 26.2

 

Table 6. Summary of Biodegradation Based on Dissolved Organic Carbon Removal (D-DOC)

Reaction

Mixtures

Blank-corrected DOC (mg/L)

Average D-doc (%)

Day 0

Day 28

Aniline

Replicate A

Replicate B

Mean

 

 

 

84.0

9.3

88.9

83.7

7.0

91.6

83.9 ± 0.2

8.2 ±1.6

90.3 ± 1.9

Piperazine

Replicate A

Replicate B

Mean

 

 

 

15.6

8.5

45.5

15.8

0.8

94.9

15.7 ± 0.1

4.7 ± 5.4

70.2 ± 34.9

 

Validity criteria fulfilled:
yes
Remarks:
But the study does report differences between piperazine replicates >20%
Interpretation of results:
inherently biodegradable
Conclusions:
The study does report differences between piperazine replicates >20% (which does not strictly meet the validity criteria).
The observed variability in the test material biodegradation rates between replicates in the study (i.e. a difference of 38.7%, 37.4%, and 49.4% biodegradation between piperazine replicates based on either biological oxygen demand, carbon dioxide evolution, or dissolved organic carbon removal, respectively) was attributed to different microbial populations between replicates, and not likely based on an inaccuracies with the dosing or study conditions, since the variability between replicates of the concurrent positive control (i.e. aniline) was low (i.e. a difference of 1.4%, 2.2%, and 2.7% biodegradation between aniline replicates based on either biological oxygen demand, carbon dioxin evolution, or dissolved organic carbon removal, respectively). Notably, one of the test material replicates had an “S” shaped curve typically observed for rapidly degrading substances. The other test material replicate had a linear increase in biodegradation over time, which was estimated to reach 60% biodegradation in ~ 31 days. The nitrate/nitrite levels differed in the final reaction vessels, also suggesting different microbial populations and pathways. The removal of dissolved organic carbon mirrored the oxygen consumption results and provides supporting evidence that there were no errors in the measurement of oxygen consumption.
The report also states there was some evidence of toxicity to the inoculum, which could have altered the microbial populations, resulting in the variation observed. The report notes that the higher replicate was more turbid, suggesting more microbial growth.
Finally, it should be considered that the likely intent of this validation criterion was to ensure adequate precision in the preparation and analysis of these biodegradation tests, and not to exclude the possibility for natural biological variability.

Based on the considerations given above that the result should be considered valid, and piperazine should be regarded as readily biodegradable.
Based on the fact that the validity criteria of West et al., 2007 is formally not met, we consider PIP as not readily biodegradable as worst-case. We therefore propose to repeat the OECD 301 study.
Executive summary:

In a ready biodegradability test according to the OECD guideline 301F - Manometric Respirometry test, O2 consumption, CO2 removal and DOC removal was monitored. The inocolum was activated sludge from primarily domestic sources. A biodegradation of 65% and 70% respectively for O2 and CO2 removal was reached, and for DOC 39% at 28 days.
Based on the fact that the validity criteria of West et al., 2007 is formally not met, we consider PIP as not readily biodegradable as worst-case.

Description of key information

Based on the fact that the validity criteria of the ready test is formally not met, we consider piperazine as not readily biodegradable as worst-case.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information

Assessment:

In order to assess the readily biodegradability of the substance a GLP study conducted according to OECD Guideline 301F is available. The degradation of the test item was determined to be 65% (O2 consumption, after 28 days, West, R.J. et al., 2007). The substance is based on the results of this OECD TG 301F considered to be readily biodegradable according to OECD criteria but the study does report differences between piperazine replicates >20%.

In addition, in a prolonged Closed Botlles test according to OECD 301D guideline, piperazine was degraded 76% (CO2 evolution) after 70 days (van Ginkel, 1990). Piperazine is found to be inherently biodegradable in a prolonged Closed Bottle Test. Furthermore, there is an inherent biodegradation study (OECD 302A) available for the substance. This GLP study assessed inherent biodegradability within 52 days, giving a degradation of 96% for the test substance (DOC removal, van Ginkel, 1992) after an initial lagtime of 30 days. In conclusion, based on the fact that the validity criteria of the ready test from West (2207) is formally not met, we consider piperazine as not readily biodegradable as worst-case.

QSAR-disclaimer

 In Article 13 of Regulation (EC) No 1907/2006, it is laid down that information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI (of the same Regulation) are met.

According to Annex XI of Regulation (EC) No 1907/2006 (Q)SAR results can be used if (1) the scientific validity of the (Q)SAR model has been established, (2) the substance falls within the applicability domain of the (Q)SAR model, (3) the results are adequate for the purpose of classification and labeling and/or risk assessment and (4) adequate and reliable documentation of the applied method is provided.

The criteria listed in Annex XI of Regulation (EC) No 1907/2006 are considered to be adequately fulfilled and, therefore, the endpoint(s) sufficiently covered and suitable for risk assessment.

Metabolites

The degradation products were assessed using the QSAR model CATALOGIC 301C v11.16 (OASIS Catalogic v5.14.1.5).

CATALOGIC 301C v11.16 (OASIS Catalogic v5.14.1.5) predicted for piperazine 8 metabolites, identifying 2 metabolites as relevant degradation products in terms of PBT/vPvB assessment, with an estimated quantity of ≥ 0.1% (for details see ‘Attached background material’ of the respective Endpoint Study Record).

All relevant metabolites were calculated to be readily biodegradable (≥ 60% after 28 days, based on BOD), thereby not fulfilling the screening criteria as potentially P/vP. All metabolites have a log Kow ≤ 3, thereby not fulfilling the screening criteria for bioaccumulation (B/vB) as laid down in Section 3.1 of REACH Annex XIII. In conclusion, all (relevant) predicted metabolites are not expected to significantly accumulate. For details see the attached file “110-85-0_The predicted relevant metabolites from the CATALOGIC 301C v11.16.pdf” in IUCLID Ch. 13.2

Based on modeled data relevant degradation products present in concentration of ≥ 0.1% (equivalent to quantity setting in OASIS CATALOGIC: ≥0.001 [mol/mol parent]) do neither fulfill the PBT criteria (not PBT) nor the vPvB criteria (not vPvB).

Hence, based on the available experimental data on the substance itself along with the modeled data for the relevant degradation products, it is considered that piperazine (CAS 110-85-0) is not readily biodegradable, according to OECD criteria.