Bronopol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

Cited as OECD Guide-line 202, part 2 (Daphnia sp., Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Initially duplicate samples from three concentrations, i . e. 0.017, 0.17 and 1.7 mg/l (the lowest, a middle and the highest) and the control were taken
for analysis. However, after the concentration of 1.7 mg/l was deleted from the range, duplicate samples were taken from the nominal concentration of 0.53 mg/l.
SAMPLING
- Frequency: at the beginning of the test (nominal day 0), every 7 days duning the exposure period and on the last day of exposure.
- Volume: 10 ml; 50 µl of acetate buffer (pH 4-4.5) was added immediately after sampling.
- Storage: the samples taken an nominal day 0 were analysed an the day of sampling. The samples taken an days 7 and 14 had been stored at -20°C until analysis an day 21, when the last samples were taken and analysed.
- Before storage at -20°C the samples were treated as follows: after acidifying the samples, they were filtered through a 0.45 µm filter to remove the algal cells. From all concentrations reserve samples were taken at the same time as the duplicate samples. These reserve samples were stored at -20°C; same reserve samples were analysed.

Vehicle:

yes

Details on test solutions:

PREPARATION OF TEST MEDIA
- For each test concentration, a stock solution was prepared in water buffered at a pH of 4. For buffering, 5 µl of acetate buffer was added per ml stock.
- The concentrations in the different stocks were 4.2, 13.4, 42, 133 and 420 mg/l, respectively.
- The computer-controlled flow-through system was programmed to dose 80 µl per 2 minutes (2.4 ml/h) of each stock in separate mixing vesseis. - - This dosing was performed by controlling a dilutor (Gilson Dilutor 401) with a syringe of 500 µl.
- In the mixing vesseis, the dosed volume of the stock was mixed with the dilutlon water, which was dosed at a flow of 20 ml per 2 minutes (600 ml/h)
- The results of pretests proved that the actual concentration of bronopol in the stocks buffered at a pH of 4 remained abaut 100% relative to the
nominal concentration for up to 72 hours. Therefore the stocks were renewed three times per week.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species: Daphnia magna STRAUS 1820

BREEDING
- Medium: dutch standard water (DSW) was formulated using Milli-Ro water according to Adema with the following composition:
Ca2+ = 54.4 mg/L
Mg2+ = 17.5 mg/L
Na+ = 27.4 mg/L
K+ = 7.8 mg/L
Cl- = 96.6 mg/L
SO4^2- = 70.1 mg/L
HC03- = 84.8 mg/L
The hardness was 11.8 °DH and the pH 8.2 +/- 0.2 after aeration.
- Milli-Ro water: tap-water purified by reverse osmosis
- Start of each batch: newborn animals, i.e. less than 3 days old; about 250 placed in 10 l of medium in an all-glass culture vessel
- Maximum age of cultures: 4 weeks
- Renewal of the cultures: after 7 days of cultivation, half of the medium is renewed twice a week
- Temperature of medium: 18 - 22°C
- Feeding: daily; suspension of freshwater algae (Chlorella pyrenoidosa)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

22 ± 0.8°C

pH:

7.8 to 8.1

Dissolved oxygen:

> 7 mg/L

Nominal and measured concentrations:

- Nominal test concentration (mg/L): 0 (control), 0.017, 0.053, 0.17, 0.53 and 1.7
- Recovery of test substance at test initiation ranged between 35 and 52%. At test ending, recovery for the 0.53 mg/l concentration was about 13%. 0.017, 0.17 and the 1.7 mg/l test concentrations were collected for analysis

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass vessel; 1.5 litre was used, containing four test containers (stainless steel mesh with mesh width of 178 µm)
- Renewal rate of test solution: preliminary studies showed that the apparent breakdown of bronopol was accelerated by the addition of algae as food source. The flow was therefore increased to the maximum practicable rate of 600 ml/h to optimize the maintenance of the bronopol concentrations as much as possible.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Feeding during test: At each renewal of the dilution water an algal suspension (Chlorella pyrenoidosa) was added as feed for the daphnia. This suspension was made by inoculating growth medium with Chlorella pyrenoidosa from a pure culture.
- Feeding rate: The amount of cells for the feeding of the daphnia was lowered to decrease the adverse influence of the algae on the bronopol concentration. During the first seven days the daphnia were fed with 10^5 cells/ml at a flow rate of 600 ml/h. In the second week, the ration was doubled, i.e. 2.10^5 cells/ml

FURTHER TEST CONDITIONS
- Photoperiod: 16 h light:8 h dark
- Light intensity: ca. 570 lux

TEST MEDIUM / WATER PARAMETERS
- Medium: Dutch Standard Water; the dilution water was mixed with the stock solution of test substance in a mixing vessel before entering the test vesseis. The dilution water was renewed daily.
- Intervals of water quality measurement: samples for analysis were collected at intervals of 7 days during the exposure period, and at test ending

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The parental daphnia were observed for immobility/mortality and presence of eggs in the brood pouch. The F1 generation was examined for number and condition of newborn, unhatched eggs and incidence of immobility.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.27 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: overall

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.27 - 0.88 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.27 - 0.88 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

21 d EC50 (parental immobility) = 0.53 - 1.7 mg/L (nominal)
21 d EC50 (reproduction) = 0.53 - 1.7 mg/L (nominal)
21 d LOEC (parental immobility) = 1.7 mg/L (nominal)
21 d NOEC (overall) = 0.53 mg/L (nominal)

- Appearance of first brood: first young were born within 7 days following the start of exposure at all concentrations, except 1.7 mg/L (nominal) where all parental daphnia died.
- Immobility among newborn: during the exposure period of 21 days no significant (> 10%) immobilization among newborn was observed at concentrations of 0.53 mg/L (nominal) or lower.
- Number of unhatched eggs: no biologically significant differences in number of unhatched eggs were observed between the concentrations tested during the reproduction phase
- No adverse effects of bronopol on the reproduction of Daphnia magna could be reported. From day 9 onwards, statistically significant stimulation of reproduction was observed at the nominal concentration of 0.53 mg/L.The cumulative mean number of living offspring per parent produced at nominally 0.53 mg/L reached a value of 1.5 times that recorded in the control.
Exposure to the other test concentrations also resulted in higher rates of reproduction as compared to the control with statistically significant differences at nominally 0.053 and 0.17 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.27 mg/L

Additional information

A 21-day Daphnia magna reproduction test under flow-through conditions with bronopol was performed according to OECD guideline 202 [Boots Pharmaceuticals 1992]. Test parameters were mortality, immobility and presence of eggs in the brood pouch of parental daphnia, as well as number and condition of newborn, unhatched eggs and incidence of immobility in the F1 generation.

At the highest concentration (1.7 mg/L), 100 % mortality was observed, whereas in all other treatments, mortality was < 20 % and therefore within the control range. The 1.7 mg/L treatment has therefore subsequently been deleted from the concentration range.

For all tested concentrations (except for the highest test concentration of 1.7 mg/L where 100 % parent mortality occured), no adverse effects on reproduction could be detected (number and condition of newborn, unhatched eggs, immobility of newborn). In fact, from day 9 of exposure a statistically significant stimulation of reproduction was noted. This increase in reproductive capacity was considered not to be an adverse effect.

Analytical monitoring of the test substance showed a recovery rate ranging from 35 to 52 % of nominal concentrations. At the end of the test, the recovery rate was about 13 %. This accelerated degradation of bronopol was probably due to a combination of factors including adsorption to algal cells, the presence of bacterial growth and the pH level of the test medium (ranging from 7.8 - 8.3). Hence, bronopol is known to hydrolyse rapidly at pH values above 5. This hydrolysis might mainly be responsible for the low analytical recovery reates. Even an increase of the flow rate did not succeed in maintaining test concentrations of bronopol.

The NOEC was determined to be 0.53 mg/L (based on nominal concentrations) and 0.27 mg/L (based on measured concentrations), respectively.