Bronopol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-JAN-21 to 2006-MARCH-07

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD 201
92/69/EEC Method C.3.
US EPA OPPTS 850.5400

Principles of method if other than guideline:

The light intensity was 4014 lux and therefore slightly below the recommended range of 4440-8880 lux. However, the increase in cell density in the control group of factor 94 within 72 hours demonstrated optimum conditions for algal growth.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytics:

For analysis of Bronopol concentration, the test medium was sampled at 0 h (fresh medium) and 96 h (from pooled replicates from each treatment and control group). All samples were analysed immediately without storage.

Algal growth:

For the determination of algal cell densities, aliquots of the test medium were collected from each replicate at approximately 24-h intervals. Samples were held for a mximum of 3 days under refrigerated conditions sufficient to inhibit growth until cell counts could be performed.

Vehicle:

no

Details on test solutions:

One stock solution was prepared for each of the 6 concentrations tested. A primary stock solution was prepared by dissolving Bronopol in freshwater algal medium at a nominal concentration of 10 mg/L. An aliquot of the primary stock solution was diluted with algal medium to prepare a secondary stock at nominal test concentration of 300 μg/L. The secondary stock was proportionally diluted with algal medium to prepare the five additional test solutions at nominal concentrations of 135, 61, 27, 12 and 5.5 μg/L. All test solutions appeared clear and colorless.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater greenalga Scenedesmus subspicatus
- Strain: CCAP 276/22
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), UK
- Age of inoculum (at test initiation):


ACCLIMATION
- Acclimation period: Algae were regularly transferred into fresh growth medium to provide suitable axenic subcultures, which were in the exponential phase of growth.
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Results are aslo available after 72 hours

Hardness:

no data

Test temperature:

23.7 – 24.3°C

pH:

At the start of the test: 7.5 – 7.7;
at the end of the test (96 h): 7.4 – 8.0

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

The nominal test concentrations were 0, 5.5, 12, 27, 61, 135 and 300 μg Bronopol/L.

The initial (0-hour) measured test concentrations were 0, 5.2, 12, 28, 63, 102 and 305 μg/L. At the end of the test period (96 hours), Bronopol concentration was below the limit of quantification in all treatment groups.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 250 mL Erlenmeyer flasks containing 100 mL of test medium
- Type (delete if not applicable): However, flasks were plugged with foam stoppers
- Aeration: Continuous shaking on mechanical shaker at 100 rpm
- Initial cells density: 1.0 x 104 cells/mL
- Control end cells density: 9.4 x 105 cells/mL at 72 h

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


GROWTH MEDIUM
- Standard medium used: yes according to OECD 201
Nominal concentrations of the nutrients in the medium were: NaHCO3 50.0 mg/L, CaCl2 x 2 H2O 18.0 mg/L, NH4CL 15.0 mg/L, MgS04 x 7 H2O 15.0 mg/L, MgCl2 x 6 H2O 12.0 mg/L, KH2PO4 1.6 mg/L, Na2EDTA x 2 H2O 0.1 mg/L, FeCl3 x 6 H2O 0.08 mg/L, MnCl2 x 4 H2O 0.415 mg/L, H3BO3 0.185 mg/L, Na2MoO4 x 2 H2O 0.007 mg/L, ZnCl2 0.003 mg/L, CoCl2 x 6 H2O 0.0015 mg/L, CuCl2 x 2 H2O 0.00001 mg/L.


TEST MEDIUM / WATER PARAMETERS
Please see above.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: Continuous light
- Light intensity and quality: Cool-white fluorescent light, 4014 lux (range: 3910 – 4390 lux)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No additional effects, e.g., changes in algal cell morphology, were found in any of the test concentrations.

Table1: Intital concentration of bronopol at the start of the test and on day 4 at test termination

Treatment [µg/L]	0 hours [µg/L]^&	96 hours [µg/L]^+	Geo. Mean [µg/L]*
Control	< LOQ (2.67)	< LOQ (2.67)	n.d.
5.22	5.22	< LOQ (2.67)	2.6
12.0	12.0	< LOQ (4.0)	4.9
27.5	27.5	< LOQ (4.0)	7.4
63.0	63.0	< LOQ (8.0)	15.9
102.0	102.0	< LOQ (16.0)	28.6
305.0	305.0	< LOQ (40.0)	78.1

& The measurements at 0 hours confirm the correct dosing of the test substance.

+ Samples collected on day 4 had recoveries that were not quantifiable.

* In addition to the reported values, geometric mean of measured concentrations of Bronopol were calculated using one-half the respective LOQ for the 96 h-data.

In regards of the decline in the test substance concentrations during the test, day 0 measured concentrations were used in the estimation of EC and NOEC values.

The observed decline of the concentration of the parent test material is in line with the known rapid hydrolysis of the product. It does not reflect a loss of test material from the test system and correction, e.g. using weighted averages is not appropriate as the break-down products formed contribute to the observed effects. The mean toxicity values based on initial measured concentrations are therefore considered as most relevant for the environmental risk assessment.

Table 2: Cell concentration data (mean of 3 replicates)

Nominal conc.		Control	5.5	12	27	61	135	300
Initial measured conc.		Control	5.2	12	28	63	102	305
Geometric mean conc.		Control	2.6	4.9	7.4	15.9	28.6	78.1
Average cell density		[μg/L]
0 d [cells/mL]		1.0 x 10^4	1.0 x 10^4	1.0 x 10^4	1.0 x 10^4	1.0 x 10^4	1.0 x 10^4	1.0 x 10^4
1 d [cells/mL]		1.75 x 10^4	1.86 x 10^4	1.72 x 10^4	1.60 x 10^4	1.23 x 10^4	1.23 x 10^4	1.35 x 10^4
2 d [cells/mL]		1.66 x 10^5	1.71 x 10^5	1.38 x 10^5	4.87 x 10^4	1.42 x 10^4	1.28 x 10^4	1.27 x 10^4
3 d [cells/mL]		9.39 x 10^5	8.12 x 10^5	5.31 x 10^5 *	8.38 x 10^4 *	1.46 x 10^4 *	1.38 x 10^4 *	1.40 x 10^4 *
4 d [cells/mL]		26.61 x 10^5	27.42 x 10^5	24.14 x 10^5	6.63 x 10^5 *	4.82 x 10^4 *	1.42 x 10^4 *	1.14 x 10^4 *
Inhibition relative to the ontrol  [%]	72h	--	14	44	91	98	99	99
	96h	--	-3.0	9.3	75	98	99	100

* statistically significant difference (p<0.05) at 72 and 96 hours from the control using Dunnett’s test

 

Table 3: Average specific growth rates and areas under the growth curves (based on daily cell counts)

Nominal conc.		Control	5.5	12	27	61	135	300
Initial measured conc.		Control	5.2	12	28	63	102	305
Geometric mean conc.	Time	Control	2.6	4.9	7.4	15.9	28.6	78.1
Growth parameter	interval	[μg/L]
Average specific growth rate [μ / h]	0 – 24 h	0.0224	0.0258	0.0223	0.0194	0.0087	0.0086	0.0123
	0 – 48 h	0.0587	0.0591	0.0547	0.0317	0.0072	0.0051	0.0049
	0 – 72 h	0.0630	0.0604	0.0551 *	0.0293 *	0.0052 *	0.0043 *	0.0046 *
	0 – 96 h	0.0581	0.0585	0.0571	0.0431 *	0.0148 *	0.0035 *	0.0013 *
Area under growth curve (AUC) [cells/mL]	0 – 24 h	89956	103456	86332	72268	28000	27980	41696
	0 – 48 h	2070920	2135972	1709812	608288	106916	89188	115528
	0 – 72 h	15112500	13692448	9492404 *	1957352 *	213560 *	168316 *	195352 *
	0 – 96 h	58071396	56099592	44582300 *	10674796 *	727896 *	264312 *	260316 *

* statistically significant difference (p<0.05) at 72 and 96 hours from the control using Dunnett’s test

Table4: Effect concentrations derived from eCA. The EC10 is the value recommended in OECD 201 to be used as endpoint. Hence, eCA has estimated statistically an EC10 value for the key study

Endpoint	Exposure		Results [mg a.s./L]
	design	duration	ErC10	ErC50	NOEC (growth rate)
Growth inhibition	Static	72 hours	0.0125 (72 h, initial measured) Model LL4	0.0265 (72 h, initial measured) Model LL4	0.0052 (72 h, initial measured)

 

 

Validity criteria fulfilled:

yes

Remarks:

The cell density in the controls increased by a factor of 94 within 3 days (1.0 x 104 cells/mL at 0 h and 9.4 x 105 cells/mL at 72 h).

Description of key information

Very toxic to aquatic algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.026 mg/L

EC50 for marine water algae:

0.178 mg/L

EC10 or NOEC for freshwater algae:

0.013 mg/L

EC10 or NOEC for marine water algae:

0.052 mg/L

Additional information

In a key study, the growth inhibition on freshwater algae has been examined in a 72 -hour static GLP guideline study according to OECD 201 [Wildlife international, Ltd., 103A-119A, 2006]  with the freshwater species Desmodesmus subspicatus. The ErC50 was 0.0265 mg/L and the ErC10 was 0.0125 mg/L of initial measured concentration.

In a key study, the growth inhibition on saltwater algae has been examined in a 72 -hour static GLP guideline study according to OECD 201 [Wildlife international, Ltd., 103A-120A, 2006]  with the saltwater species Skeletonema costatum. The ErC50 was 0.178 mg/L and the NOErC was 0.052 mg/L of nominal concentration.

Supporting studies are summarised below

The growth inhibition on algae has been examined in a 72 -hour static GLP guideline study according to ISO 10253 [Knoll MicroCheck 1998 (a)] with the saltwater species Skeletonema costatum. Additionally, algistatic and algicidal effects were investigated according to US EPA OPPTS 850.5400 "Algal Toxicity", Tiers I and II. At test termination for the growth inhibition examination, an ErC50 = 0.25 mg/L (nominal) and a NOEC = 0.08 mg/L (nominal) could be determined. Algistatic effects were seen at 0.42 mg/L (nominal) and algicidal effects were observed at 1.0 mg/L (nominal).

The toxicity of bronopol to freshwater algae species has been investigated in an OECD 201 guideline study with Selenastrum capricornutum as test organism [Boots Microcheck 1994]. After the exposure period of 72 h an ErC50 of 0.37 mg/L and a NOErC of 0.1 mg/L was determined.

Further on, two growth inhibition tests on algae were carried out according to OECD guideline 201 [Boots Microcheck 1994] with two different freshwater algae species. The 72 -hour ErC50 was determined to be in the range of 0.89 to 2.84 mg/L for Chlorella vulgaris and > 1.00 mg/L for Scenedesmus subspicatus. The NOErCs were determined to be 0.32 and 0.10 mg/L for Chlorella vulgaris and Scenedesmus subspicatus, respectively. All results refer to nominal concentrations since no clear quantification of the actual exposure concentrations could be achieved. The inconsistent results obtained by analytical monitoring may be related to the fact that Bronopol rapidly hydrolyses at pH values ranging from 7 to 9.

Additionally an algae growth inhibition study with Scenedesmus subspicatus was conducted according to OECD guideline 201 [Knoll MicroCheck 1998 (b)]. Additionally, algistatic and algicidal effects were investigated according to US EPA OPPTS 850.5400 "Algal Toxicity", Tiers I and II. At test termination after 72 h, the ErC50 for Bronopol was determined to be 0.67 mg/L (nominal) and the NOErC was determined to be 0.1 mg/L (nominal). An algicidal effect of Bronopol after 7 days was indicated at 3.2 mg/L and higher test concentrations.

The acute and chronic results obtained from these studies were similar to the ones retrieved from the key study and can therefore be used as supporting evidence of the acute toxicity of Bronopol to aquatic algae.