Bis(2-ethylhexyl) phthalate

Biodegradation in water and sediment: simulation tests

Key value for chemical safety assessment

Half-life in freshwater:

50 d

at the temperature of:

12 °C

Half-life in freshwater sediment:

300 d

at the temperature of:

12 °C

Additional information

 Simulation tests in surface water:

Four studies on degradation of DEHP in surface water are available.

-Subba-Rao et al. (1982) investigated the mineralisation of 14C-DEHP in two different filtrated lake waters (eutrophic and oligotrophic). Test concentrations were 0.02, 0.2, 2.0 and 200μg/l. The incubation flasks were kept in the dark at 29°C without shaking. Samples were taken at intervals for 40 and 60 days, respectively. Generated 14CO2 was removed and the remaining radioactivity in the solutions was measured. After 40 days of incubation, 35 – 71% of the original DEHP was mineralised in the eutrophic lake water. The mineralisation half-lives in eutrophic water could be calculated to approximately 22-64 days at 29°C based on first order kinetics. No detectable lag-phase was observed, and the mineralisation did not seem to be concentration-dependent. In the oligotrophic lake water, no mineralisation occurred during the 60 days of incubation. As it is the only mineralization study and it is well described this study is considered as reliable with acceptable restriction and is selected as key study.

The three following references studied primary degradation of DEHP in different surface waters.

-Saeger and Tucker (1976) studied the transformation (primary degradation) of DEHP in river water from Mississippi. Test concentration was 1 mg DEHP/l in 200 ml portions of river water. The bottles were kept at room temperature. Samples were taken after 7, 14, 28 and 35 days for analysis by means of GC-FID. After 35 days, 35% of the original DEHP was unaltered. A sterile control was included to account for possible substance decline caused by other factors than biodegradation. Thus, with a degradation of 65% within 35 days an approximate DT50 of 30 days can be estimated (20°C).

-Ritsema et al (1989) studied the biotransformation (primary degradation) of DEHP at 4°C and 20°C in river water from Rhine. Test concentration was 3.3μg/l. Samples were taken on 0, 1, 3, 7 and 10 days after the start and were analysed by means of GC-ECD. Recovery of the method of analysis was 83 – 97%. During the 10 days of incubation, no degradation occurred at 4°C, while approximately 33% of the parent compound was transformed at 20°C within 10 days. From these data, an approximate DT50 of 15 days can be estimated (20°C).

-Furtmann, (1993) investigated the degradation of DEHP and other phthalates in Rhine water. The background concentration in the water was approximately 0.4μg/L and the samples were amended to concentrations of 2 and 5μg/L, respectively. Initially, degradation was rapid (at 20°C), with 90% primarily degraded after 8 days. However, at a concentration close to the background, a threshold level appears to have been reached, below which no further degradation was observed. In parallel samples kept at 4°C, no degradation occurred during the incubation period. Tanking the data obtained at 20 °C, an approximate DT50 of 4.4 days can be estimated (20°C).

These three studies are reliable with acceptable restrictions. However, since the main metabolite MEHP (as identified in several studies) is ecotoxicologically relevant and considered even more toxic than the parent compound, the estimation of the biodegradation rate should preferably not be based on primary degradation. Therefore these studies are regarded as supporting studies.

From the study of Subba-Rao et al. (1982) it appears that the trophic status of the surface water play a role in the degradation of the DEHP: While degradation of DEHP was observed in eutrophic water (35 – 71% was mineralised at 29°C after 40 days), in oligotrophic water no mineralisation was observed during 60 days. Moreover the results of two studies indicate that the degradation rate is highly temperature-dependent since no degradation of DEHP took place at 4°C (Ritsema et al., 1989; Furtmann, 1993) while considerable degradation was observed at higher temperatures of about 20°C (appr. 65% degraded within 35 days according to Saeger and Tucker, 1976; appr. 33% within 10 days according to Ritsema et al., 1989; appr. 90% within 8 days according to Furtmann, 1993) and 29°C (up to 71% within 40 days according to Subba-Rao et al., 1982).

According to the guidance on information requirements and chemical safety assessment - chapter R16: Environmental exposure estimation (ECHA 2008), the half-life in surface water should be based on simulation tests. Considering however the data variability in these tests probably due to the different test conditions applied, it seems preferable in a realistic worst case approach to base the half-live of DEHP in surface waters on results of screening test on biodegradability according to Table R16 -7. If so, degradation rate based on Diefenbach (1994) would set a half-life of 15 days in surface water. However, considering the variability of the degradations observed in test simulating natural conditions, we assume that a more severe value is preferable. In conclusion, a half-life for DEHP in surface waters of 50 days (at 12 °C) for readily biodegradable substances failing the 10 day window is applied for the CSA.This is a conservative approach taking due account of data pointing to lower degradation rates at lower temperatures and being in line with EU-RAR (2008).

Simulation tests in sediment:

In total, four studies are available investigating degradation of DEHP in sediments. In aerobic waters, sediment is generally composed of a thin aerobic surface layer and predominantly anoxic lower layers. The highly reliable key study (Kickham, 2010; RL 2 solely due to not being compliant to GLP) performed similar to OECD 308 takes this into account: Marine sediment plus overlying bottom water was sampled from False Creek embayment (downtown Vancouver, British Columbia) from a depth of approximately 5 m. Sediment was spiked with DEHP, was sampled into 125 ml glass jars with metal lids (30g of spiked sediments and 10 mL of overlying False Creek water) and left essentially undisturbed for 144 days incubation time at 13 °C. To allow for headspace gas exchange, jars were opened 2 times per week under sterile conditions, followed by gently swirling at 120 rpm in a rotary shaker for 5 minutes. Dissolved oxygen concentration was between 1 and 2 mg/L (water) during the first days of incubation (below the target value) and rose slowly until day 70 to approximately 5 mg/L, which was approximately constant till the end of the experiment. Extensive quality assurance steps were taken to ascertain that observed decline in test item concentration was actually due to biodegradation by microorganisms. Concentrations of DEHP declined slowly in test sediment over the incubation period. Approximately 85% of the original amount of chemical remained after 144 days. Applying first order kinetics, the following degradation half-life for DEHP in natural marine bulk sediment (aerobic surface layer, anoxic lower layers) at 13 deg C (incubation period 144 days) was determined:

DT50 [d] = 337 days

The DEHP metabolite Mono-2-ethylhexyl phthalate (MEHP) was detected above the limit of quantification, and it slowly increased from 48 to 162 ng/g dw over the first 12 days of incubation before declining and stabilizing at 25-30 ng/g dw for the remainder of the incubation. This confirmed the slow (but statistically significant) biodegradation rate determined for DEHP. Primary biodegradation of MEHP was demonstrated to be very rapid in False Creek sediments (t1/2 of 26 +/- 9 hours at 22 deg C). Thus, the continuous input from DEHP degradation must have led to amounts of MEHP above LOQ. However, MEHP was shown not to accumulate in sediment but rather to be efficiently degraded itself.

These results are supported by a reliable (with restrictions, RL 2) study performed by Chang et al. (2005).Using sediment from a highly contaminated river of Taiwan together with synthetic medium similar to the one described in OECD 311, thorough degradation of diethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) within 84 days underanaerobic conditionsat 30°C and pH 7 could be demonstrated. A decrease in temperature from 30 to 20 deg C led to an increase in half-life, while addition of yeast extract or detergents at 1 CMC decreased observed half-lives for DEHP. Methane production and negative control values demonstrated biological activity of the system, as did the successful isolation of four single bacterial strains able to degrade mixtures of DEP, DBP and DEHP under anaerobic conditions. Half-life was higher with DEHP alone compared to a triple mixture of DEHP, DEP and DBP under otherwise identical conditions. The following decisive half-life foranaerobic degradation(first order kinetics) is reported for DEHP alone:

DT50 (30°C, pH 7): 33 days

This much lower half-life compared to the study of Kickham (2010) may be explained by the use of synthetic medium instead of natural water from the sampling site, pre-adapted microorganisms due to the heavy pollution of the river, the considerably higher temperature applied as well as freshwater conditions, which may be more favorable for degradation. With temperature correction according to R.16 guidance, a DT50 (12°C, temperature adapted) of 139.3 days results, which is still considerably lower than the half-life determined for marine sediment.

By weight of evidence from these two studies, a conservative half-life (DT50) for DEHP of 300 days for bulk sediment (aerobic + anaerobic) is derived and used for chemical safety assessment, valid at the relevant temperature of 12 degree C.

This value of DT50 (12°C, bulk sediment) for DEHP is supported by further two studies: Firstly the study of Johnson and Lulves (1975), who determined an aerobic mineralization of DEHP in sediment from a freshwater pond (no synthetic medium but natural water) at 22 °C of approximately 60% within 28 days, corresponding to a roughly estimated DT50 of 23.3 days at 22°C and transformed to 12 °C of 52 days under aerobic conditions. And secondly the work of Johnson et al (1984), who determined similar degradation levels for DEHP (radiolabelled) in freshwater sediments under aerobic (14% within 28 days at 22°C) and anaerobic (9.9% within 28 days at 22°C) conditions. Extrapolating the degradation level under anaerobiosis, an approximate DT50 (22 °C) of 141 d results which, after transformation to 12 °C according to R.16 corresponds to DT50 (12°C, temperature adapted) of 314 days, which fits very well to the above derived value of 300 days for total sediment.

In conclusion, the available new data (Kickham, 2010; Chang et al., 2005) justify to deviate from EU-RAR (2008) for DEHP, where a DT50 for aerobic sediment of 300 and an infinite DT50 for anaerobic sediment was assumed, which led to a DT50 for bulk sediment (sediment overall) of 3000 days.