Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 1998 - 02 March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

1 L obtained from Haddington Municipal Sewage Works and used on the day of collection. On receipt, the sludge was shaken by hand then added to a blender (low; ca. 2 mins). The solution was decanted and left to settle into to form an aqueous and solid layer. The aqueous layer was used to innoculate the bioreactor vessels.

Duration of test (contact time):

29 d

Initial conc.:

ca. 10 - ca. 20 other: mg OC/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

1564 mL of Milli-Q water was added to 7 2 L bioreactor flasks.20 mL of mineral media A, 2 mL of mineral media B-D (see table below) and 20 mL of activated sludge was added to each of the bioreactor vessels.The bioreactors were connected to an CO2-free air supply and purged overnight to remove excess CO2 from the system. During the incubation period the air flow was adjusted to ca. 20-100 mL/min.Aliquots, ca. 28.62 and 57.24 mg, of TBE were weighed in duplicate and added to separate bioreactor vessels to achieve concetrtion of ca. 10 and ca. 20 mg OC/L, respectively. The bioreactors were made up to 2 L by addition of 400 mL Milli-Q water.Reference and toxicity bioreactors were prepared as follows;Sodium benzoate (ca. 343 mg) was accurately weighed into a 100 mL volumetric flask which was brought to volume with Milli-Q water. A reference bioreactor was prepared by dispensing 20 mL of the sodium benzoate stock solution and 380 mL of Milli-Q water into a bioreactor to achieve a concetraion of ca. 20 mg DOC/L. A toxicity control was prepared by adding ca. 57.24 mg of TBE (equivelant to ca. 20 mg OC/L), 20 mL of sodium benzoate stock solution and 380 mL of Milli-Q water to a bioreactor flask.The final (7th) bioreactor flask was used as a blank control and contained 400 mL Milli-Q water.Each bioreactor flask was connected to a series of three 0.0125 M barium hydroxide (BaOH) traps.Sampling occured on 2, 4, 6, 8, 10, 15, 20, 25 and 29 DAT (Days After Treatment) by removing the BaOH trap closest to the bioreactor flask. The remaing BaOH traps were moved 1 position closer to the bioreactor flasks. A fresh BaOH trap was added to the end of the trap series (furthest from the bioreactor flask). Sampled traps were titrated against hydrochloric acid (HCl) as described in the analytical methods sections.Evolved CO2 and % biodegradation were calculated based on the results of the titrations, as described in the analytical methods section.

Reference substance:

other: sodium benzoate

Key result

Parameter:

% degradation (CO2 evolution)

Value:

>= 39.89 - <= 40.96

Sampling time:

29 d

Remarks on result:

other: Not readily biodegradable

Details on results:

The results from the test material bioreactors indicate an initial lag phase followed by initaition of biodegrdation. The biodegradation, expressed as a percentage of the actual versus the theoretical quantities of CO2 evolved, was 39.89 and 40.96 % after 29 days for the ca. 10 and 20 mg OC/L samples, respectively.

Results with reference substance:

The CO2 produced from the reference inoculum was 53.35 mg CO2/L, which is equivalent to 144.94 % biodegradation after 29 days.The results of the toxicity control test in which the reference item and test material were mixed was calculated on the basis of carbon available through addition of sodium benzoate, the reference item. Biodegrdation of 202.91 % was noted after 29 days, indicating that TBE, at a concetration of 28.74 mg/L, did not have an inhibitory effect on the microbial population.

Table 1       CO₂ Evolution of TBE

Timepoint (Days)	CO2Evolved (mg CO2/2L)
	Endogenous CO2	10 mg DOC*	20 mg DOC*	10 mg DOC Cumulative	20 mg DOC Cumulative
0	0	0	0	0	0
2	3.52	0.00	0.00	0.00	0.00
4	5.28	0.00	0.00	0.00	0.00
6	4.73	1.27	6.16	1.27	6.16
8	2.31	3.80	8.36	5.07	14.52
10	1.87	1.05	3.47	6.12	17.99
15	2.75	5.94	8.75	12.06	26.74
20	2.97	1.76	2.81	13.82	29.55
25	2.31	0.50	0.77	14.32	30.32
29	0.99	0.17	0.00	14.49	30.32

* mg CO₂ in sample - endogenous mg CO₂ (from blank control)

Table 2       Biodegradation of TBE

Timepoint (Days)	Mean % Biodegradation		Mean Cumulative % Biodegradation
	10 mg OC/L*	20 mg OC/L*	10 mg OC/L	20 mg OC/L
0	0	0	0	0
2	0.00	0.00	0.00	0.00
4	0.00	0.00	0.00	0.00
6	3.50	8.32	3.50	8.32
8	10.46	11.29	13.96	19.61
10	2.89	4.69	16.85	24.30
15	16.35	11.82	33.20	36.12
20	4.84	3.80	38.04	39.92
25	1.38	1.04	39.42	40.96
29	0.47	0.00	39.89	40.96

* ((mg CO₂ produced / (ThCO₂ x mg carbon available from TBE)) x 100

ThCO₂ = theoretical CO₂ produced (3.67)

Table 3       Biodegradation of Reference Item, Sodium Benzoate

Timepoint (Days)	mg CO2Produced/2L^§	Cumulative mg CO2/2L	% Biodegradation*	Cumulative % Biodegradation
0	0	0	0	0
2	34.76	34.76	47.22	47.22
4	32.23	66.99	43.78	91.00
6	21.78	88.77	29.58	120.58
8	8.03	96.80	10.91	131.49
10	0.00	96.80	0.00	131.49
15	8.03	104.83	10.91	142.40
20	0.00	104.83	0.00	142.40
25	1.87	106.70	2.54	144.94
29	0.00	106.70	0.00	144.94

^§ mg CO₂ in sample - endogenous mg CO₂

* ((mg CO₂ produced / (ThCO₂ x mg carbon available from sodium benzoate)) x 100

ThCO₂ = theoretical CO₂ produced (3.67)

Table 4       Biodegradation of Toxicity Control

Timepoint (Days)	mg CO2Produced/2L^§	Cumulative mg CO2/2L	% Biodegradation*	Cumulative % Biodegradation
0	0	0	0	0
2	28.93	28.93	39.30	39.30
4	30.69	59.62	41.69	80.99
6	24.53	84.15	33.32	114.31
8	13.09	97.24	17.78	132.09
10	1.32	98.56	1.79	133.88
15	29.04	127.60	39.45	173.33
20	14.52	142.12	19.72	193.05
25	5.06	147.18	6.87	199.92
29	2.20	149.38	2.99	202.91

^§mg CO₂in sample - endogenous mg CO₂

* ((mg CO₂produced / (ThCO₂x mg carbon available from sodium benzoate)) x 100

ThCO₂= theoretical CO₂produced (3.67)

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

TBE was determined not to be readily biodegradable at concetrations of ca. 10 and 20 mg OC/L according to the conditions outlined in OECD Guideline 301B.TBE was found to be non-inhibitory to the microbial population contained in the sludge inoculum at a concetration of 28.74 mg/L.

Executive summary:

Ready biodegradability testing of TBE was carried out with reference to the Modified Sturm Test which is described in the OECD Guideline 301 (1992).

The mean percentage biodegrability values of TBE after the 29 -day test period were 39.89 % and 40.96 % for the 10 mg organic carbon (OC/L) and 20 mg OC/L bioreactors, respectively. A toxicity control showed that TBE was not inhibitory to the bacteria in activated sludge at a concetration of ca. 28.74 mg/L.

In accordance with the OECD 301B Guideline, it is determined that TBE is not readily biodegradable.

Description of key information

Not readily biodegradable; OECD 301B, 1992 (GLP); Hogg and Scott (1999)

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

The ready biodegradability of TBE was determined in a GLP study in accordance with OECD Guideline 301B. TBE was determined to be "not readily biodegradable" under the conditions of the test (Hogg and Scott, 1999).