Metformin hydrochloride

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July - 06 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 219 (Sediment-Water Chironomid Toxicity Test Using Spiked Water)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The total radioactivity in water and sediment of test vessels treated with 14C-Metformin at different concentrations was determined by Liquid Scintillation Counting (LSC). Furthermore, the concentrations and stability of 14C-Metformin in the application solutions, water phases and sediment extracts was determined by HPLC (High Performance Liquid Chromatography).

Before the test item application, samples were taken from the application solutions. For the determination of the stability of the test item in the application solutions, a sample was taken also immediately after the application from the application solution of the highest test concentration.

The analytical samples from Day 0 and 7 were taken from test beakers, prepared exclusively for analytical measurements in parallel to the biological test (two test beakers were prepared for each sampled test concentration, and one for the control). One of the two treated test beakers and the control beaker were sampled on Day 0. The other treated replicates were incubated under the conditions of the test until sampling on Day 7. To these additional replicates test organisms were inserted and food was added as in the biological part of the study. On Day 28, analytical samples were taken from the test beakers in the biological test.

On each sampling date, the water samples were taken from the water column without previously mixing the water. For the determination of the test item concentrations in the water and sediment, the water column was siphoned from the test beaker. Additionally, water was obtained by centrifugation of the wet sediment at about 2800 rpm for 10 minutes. Then the sediment sample was taken for analysis.

Vehicle:

no

Details on sediment and application:

An artificial (formulated) sediment was used according to the test guideline. The artificial sediment was prepared on the basis of dry weights as follows:

- Sphagnum peat: 5% (air dried, very finely ground to ≦1 mm) organic carbon content 47.3%)
- Kaolin clay (content of Al2O3: 35.0%): 20%
- Sand (Sihelco 36): 75%
- Calcium carbonate (CaCO3): 0.29%

The total organic carbon (TOC) in this artificial sediment was 2.5% (based on dry weight). All dry constituents were weighed to the correct portions. The finely ground peat was moistened with purified water by intense mixing using an ultra-turrax and gentle stirring for 2 days at room temperature in the dark. The pH of the peat suspension was 5.6 at start of moistening. CaCO3 was added for pH adjustment, and after the moistening procedure the pH was 6.1. Then all constituents were thoroughly blended for 5 min. in a HOBART laboratory mixer to receive homogeneous wet sediment. Purified water was added to obtain sufficient moisture of the final mixture (46%, based on dry weight). A sediment sample of about 10 g was shaken with 25 mL of a KCl solution (1 mol/L) for 30 minutes and then the pH was measured in this suspension. The pH of the final mixture of sediment was 6.9.

The artificial sediment was checked for absence of chemical contamination. The following parameters were analyzed in a representative sample of the artificial sediment: heavy metals (cadmium, arsenic, lead, mercury and copper), pesticides (lindane, heptachlor, malathion, total DDT, dieldrin), and PCBs (28, 52, 101, 138, 153, 180).

Preparation of the Test Vessels
The wet artificial sediment was filled into each test vessel at a layer of approximately 1.5 cm depth. This amount corresponded to 130 g wet weight with 46% water content (or 89 g dry sediment). Before adding the test water, the sediment surface was covered with a plastic plate, which floated as the water was poured onto it. Then 250 mL of test water were poured into each beaker very slowly, taking care not to disturb the sediment. The total water volume per beaker corresponded to a water column of 6 cm depth. Thus, the ratio of the depth of the sediment layer to the depth of the overlying water was 1:4. After filling the vessels with water, the plastic plate was removed. The vessels were prepared seven days before inserting the test animals, and were incubated during this period under the conditions of the test for conditioning of the water-sediment systems. The water level was marked outside on the test vessels. Water levels did not change by more than 10% during the test period. Purified water was filled up to the normal water level (approx. 15 ml per beaker) on Day 21.

Test organisms (species):

Chironomus riparius

Details on test organisms:

The study was performed with larvae of the midge Chironomus riparius bred at Harlan Laboratories Ltd..The breeding was conducted under similar test conditions (temperature, light, test water) as used in the test. Only fresh egg masses were used as source for the test animals. At the date when the test animals were placed into the test beakers, the larvae were 2–3 days old (first-instar larvae). The midge Chironomus riparius is a preferred freshwater aquatic insect species used to evaluate the toxicity of chemicals present in sediments. The test method and the test species are recommended by the international test guidelines.

Seven days before inserting the larvae into the test beakers, some fresh egg masses were taken from the test organism culture and deposited into small vessels in test water with a small amount of food (mixture of fresh green algae Scenedesmus subspicatus from a laboratory culture and a Tetra Min® fish food suspension).
20 larvae of the first larval stage (2-3 days old) were allocated randomly to each test vessel by means of a suitable pipette (four collectives of five larvae each, per vessel). When adding the larvae and also for the following 24 hours, the aeration of the water in the test vessels was stopped.
One day after adding the larvae, the test item was applied to the water column of the water-sediment systems. This day of application of the test item is defined as Day 0 of the study.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

200 mg/L as CaCO3

Test temperature:

19.5-20.6°C

pH:

7.9 ± 0.3

Dissolved oxygen:

saturated >7.5 mg/L

Nominal and measured concentrations:

32.25, 62.5, 125, 250, 500 and 1000 mg/L

Details on test conditions:

Test Vessels
Glass beakers (600 mL, approximately 8 cm in diameter) were used as test vessels. Each test beaker was covered with a lid containing a mosquito net to prevent the escape of emerged adult test animals. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

Water temperature: 19.5-20.6 °C during the experiment. The water temperature differed by less than 1.0 °C between beakers at any time during the test.
Light conditions: A 16-hour light to 8-hour dark photoperiod with a 30 minute transition period between light and darkness. Light intensity during the light period was within the range of approximately 570–860 Lux (measured approximately at water surfaces of the test beakers).
Test duration: 28 days after application of the test item (4 days after emergence of the last test animal in the control).
Aeration: During the whole study (with exception of the period from insertion of the larvae until immediately after application of the test item) the water in the water-sediment systems was gently aerated through a glass Pasteur pipette, fixed above the sediment layer.

Four replicates (test beakers) were tested in the biological test at each test concentration and in the control. Additional replicates were prepared in parallel for the analytical requirements.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

emergence rate

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

31.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

development rate

Details on results:

Analytical Results
The measured test item concentrations in the test media one hour after application corresponded well with the initial nominal test concentrations. The variability between the analytical results of the parallel test vessels per treatment was very low. Therefore, all reported biological results are related to the nominal initial concentrations of 14C-Metformin in the water column. At the two tested concentrations of 125 and 1000 mg/L, the radioactive residues in the overlaying water columns had decreased nearly constantly from about 94% of the nominal values after application to about 48-57% of the initial values at the test termination after 28 days. The radioactivity in the measured sediments continuously increased during the test period. The amount of non-extractable radioactivity in the sediment continously increased from 3% at test start to 15-36% of all found radioactivity in the sediment at test termination in the two measured concentrations.

Biological Results - Emergence Ratio
The emergence ratios per vessel in the control ranged from 85 to 100 % (thus fulfilling the guideline validity criterion of ≥70%). Up to and including the nominal test concentration of initial 125 mg/L, the mean emergence ratios of pooled sexes were not significantly lower than in the control (results of a Williams t-test with arcsin- transformed emergence rates, alpha = 0.05, one-sided smaller). From the nominal test concentration of initial 250 mg/L on, the mean emergence ratios were statistically significantly reduced compared to the control (results of a Williams t-test with the arcsin-transformed emergence rates, alpha = 0.05, one-sided smaller). The 28-day EC15 for the arcsin-transformed emergence rate of pooled sexes was calculated to be 253 mg/L (95% confidence limits: 232 – 272 mg/L). The 28-day EC50 was determined to be 322 mg/L (95% confidence limits: 301 – 346 mg/L). The sex of the small inserted larvae at the start of the test can not be differentiated. Thus, a separated emergence ratio for males and females can not be determined, and is, therefore, not taken into account.

Biological Results - Development Rate
72 out of 73 midges in the control had emerged between Days 12 and 23 (and thus fulfilled the validity criterion of the test guideline requesting the emergence not earlier than on Day 12 and not later than on Day 23). Only one female in replicate one of the control emerged on Day 24. This aspect is not considered to have an influence on the outcome of the study, since statistical analysis with and without the one too late emerged female revealed no differences in determination of NOEC/LOEC. For the lowest test concentration of initial 31.25 mg/L, the mean development rates of males and females were not statistically significantly lower than in the control (results of a Williams t-test, alpha = 0.05, one-sided smaller). At all higher nominal test concentrations of initial 62.5 to 1000 mg/L, the mean development rates of both males and females were statistically significantly reduced compared to the control (results of a Williams t-test, alpha = 0.05, one-sided smaller). The 28-day EC15 and EC50 for the development rate of males was calculated to be 88 mg/L (95% confidence limits 23 – 144 mg/L) and 601 mg/L (95% confidence limits 371 – 2147 mg/L). The 28-day EC15 and EC50 for the development rate of females was calculated to be 106 mg/L (95% confidence limits 56 – 142 mg/L) and 519 mg/L (95% confidence limits 329 – 1921 mg/L).

Biological Results - Symptoms of Toxicity
No symptoms of toxicity were observed at the larvae, pupae and emerged midges during the study.

Water parameters
During the test period, the pH values in the test media ranged from pH 7.7 to 8.5. The dissolved oxygen concentrations were at least 7.1 mg/L (= 84% oxygen saturation value) and thus sufficiently high throughout the test period. The water temperature varied between 19.5 and 20.6 °C and was thus sufficiently constant. The water temperature differed by less than1.0 °C between all beakers at any time during the study.

Appearance of the Test Media
No anomalies or significant differences between the water-sediment systems of the control and treatments were observed during the study period. Starting from Day 7, a typical slight turbidity was recorded in all test media. Furthermore, a thin film of microorganisms was observed on the walls of all test beakers including the control starting from Day 14 until test termination, and from Day 21 onwards, slight algal growth was observed on the walls of all test vessels up to and including the test item concentration of 250 mg/L . In the test item concentration of 500 mg/L, algal growth could only be observed at Day 28, while in the highest test item concentration of 1000 mg/L no algal growth was observed throughout the whole test duration.

Validity criteria fulfilled:

yes

Conclusions:

Thus, the overall 28-day NOEC of 14C-Metformin for Chironomus riparius in this water-sediment study was 31.25 mg/L. The overall 28-day LOEC was 62.5 mg/L due to a statistically significantly reduced development rate of the larvae.

Executive summary:

Study Design

Toxic effects of the test item¹⁴C-Metformin on the development of sediment-dwelling larvae of the midge Chironomus riparius in water-sediment systems were investigated following the OECD Guideline 219 “Sediment-Water Chironomid Toxicity Test Using Spiked Water” (2004).
First-instar larvae of Chironomus riparius were exposed for a period of 28 days until full maturation of the larvae to adult midges. The test parameters of the study were development time/rate of the midges and emergence ratio as the number of fully emerged male and female midges. The radiolabeled test item was applied on or into the water column in static water-sediment systems. The nominal initial test item concentrations in the overlaying water columns were 32.25, 62.5, 125, 250, 500 and 1000 mg/L. A control (water-sediment system without test item application) was tested in parallel.

Results

The analytically determined test item concentrations in the test media one hour after application corresponded well with the initial nominal test concentrations, showing a stable application solution of the highest test concentration and an analytical recovery of 94% of the test item in the median and highest test concentration. Therefore, all reported biological results are related to the nominal initial concentrations of the test item in the water column. At all test concentrations the radioactive residue in the overlaying water columns decreased nearly constantly from 94% of the nominal values after application to 48-57% of the initial values at the test termination after 28 days. The radioactivity in the sediments continuously increased during the test period. The amount of non-extractable radioactivity in the sediment continously increased from about 3% at test start to 15-36% of all measured radioactivity in the sediment at test termination in all measured concentrations.

Conclusion

Thus, the overall 28-day NOEC of ¹⁴C-Metformin for Chironomus riparius in this water-sediment study was 31.25 mg/L. The overall 28-day LOEC was 62.5 mg/L due to a statistically significantly reduced development rate of the larvae.

Description of key information

Thus, the overall 28-day NOEC of 14C-Metformin for Chironomus riparius in this water-sediment study was 31.25 mg/L. The overall 28-day LOEC was 62.5 mg/L due to a statistically significantly reduced development rate of the larvae.